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Cited by 3,999Autophagy in Disease and TherapyCalcium signaling and nucleotide metabolismCellular transport and secretion

Guidelines for the use and interpretation of assays for monitoring autophagy

Open access
Klionsky, Daniel J., Abdalla, Fabio C., Abeliovich, Hagai, Abraham, Robert T., Acevedo-Arozena, Abraham, Adeli, Khosrow, Agholme, Lotta, Agnello, Maria +1262 more·2012·Autophagy
DOI 10.4161/auto.19496PMC PMC3404883
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Abstract

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

AI snapshot

Written from a shortened version of the paper’s full text (it is longer than we read).

TL;DR

This 2012 consensus guideline updates the 2008 autophagy assay guidelines, emphasizing that autophagosome accumulation must be distinguished from autophagic flux and that no single assay is universally appropriate. It recommends using multiple complementary assays and provides method-specific cautions for monitoring macroautophagy and related processes across organisms.

Key findings

  • Autophagosome accumulation can reflect either increased autophagy induction or a block in autophagosome turnover; therefore, LC3-II or GFP-LC3 puncta alone are not measures of autophagic flux and should be complemented by flux assays.
  • Atg8/LC3 detection: LC3-I is approximately 18 kDa and LC3-II approximately 16 kDa; in yeast, Atg8 amounts increase at least 10-fold when autophagy is induced. LC3-II turnover can be assessed by comparing LC3-II levels in the presence and absence of lysosomal inhibitors, with bafilomycin A1 treatment typically kept short (1–2 h).
  • TEM can monitor autophagic structures, but reliable quantification requires volumetric morphometry/stereology in at least 20 cell profiles per sample; autophagosomes have a double membrane, whereas autolysosomes usually have a single limiting membrane.
  • The guidelines discourage relying on acidotropic dyes such as monodansylcadaverine as sufficient autophagy markers, advise against expressions like “percent autophagy,” and stress that no individual assay is guaranteed to be appropriate in every situation.

Methods

  • Design: consensus guidelines/review updating the 2008 autophagy guidelines; not a primary experimental study.
  • Data or sample: no primary sample or dataset; the paper synthesizes literature and expert recommendations across organisms, cell types, and experimental systems.
  • Main analysis: narrative, method-by-method evaluation of autophagy assays with cautionary notes and conclusions; no statistical analysis is reported.

Limitations

  • Stated: there are no absolute criteria for determining autophagic status applicable in every biological or experimental context; some assays are inappropriate, problematic, or may not work in particular cells, tissues, or organisms, and the guidelines are likely to evolve as methodologies change.
  • Stated: the guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend on the question being asked and the system being used; no individual assay is guaranteed to be the most appropriate in every situation.
  • Evident from the text: as a guideline/review rather than a primary validation study, many recommendations rely on cited literature and expert consensus and may not have been empirically tested in every system.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.4161/auto.19496

Cites

896 of 896 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

158 of 315 cited sources checked · 63 supported · 8 flagged · accuracy not yet measured

  • Premisecitation

    Basal, induced and inhibited formation of WIPI puncta closely correlate with both the protein level of LC3-II and the formation of GFP-LC3 puncta.

    “Basal, induced and inhibited formation of WIPI puncta closely correlate with both the protein level of LC3-II and the formation of GFP-LC3 puncta.419,421”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [419] source
    • cites [421] source

      The Results report that resveratrol increased LC3-II and GFP-LC3 puncta, while Figure 2 and the accompanying results report no induced WIPI-1 puncta. The paper thus gives a direct exception to the claimed close correlation, although WIPI-1 puncta and LC3-II increased together under nutrient starvation and rapamycin treatment. Page numbers are not available in our copy. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

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  • Premisecitation

    On induction of autophagy by various stimuli, the presence of BECN1- and PIK3C3-positive macroaggregates can be detected in the region of the Golgi complex by immunofluorescence.

    “On induction of autophagy by various stimuli the presence of BECN1-and PIK3C3positive macroaggregates can be detected in the region of the Golgi complex by immunofluorescence.115,435”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [115] source

      In “Both PINK1 and Beclin1 localize to the mitochondrial compartment” (page unavailable), the authors report that they failed to identify Beclin1 co-localization with Golgi markers. The paper discusses autophagy induction, but our copy does not report BECN1- and PIK3C3-positive macroaggregates near the Golgi under those conditions. (LLM, conf=0.79)

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    • cites [435] source
  • Premisecitation

    After sufficient mitochondria proliferation, shifting the cells back to a fermentable carbon source such as glucose will cause the autophagic degradation of superfluous mitochondria.

    “After sufficient mitochondria proliferation, shifting the cells back to a fermentable carbon source such as glucose will cause the autophagic degradation of superfluous mitochondria.497”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [497] source

      In the mitophagy-monitoring results (page not provided), cells grown in lactate medium and shifted to glucose medium showed barely detectable mitophagy. The authors conclude that shifting to a fermentable carbon source alone did not induce detectable mitophagy; they detected it when the shift also involved nitrogen starvation. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

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  • Study design and conductcitation

    TEM is a valid and important method both for qualitative and quantitative analysis of changes in various autophagic structures that sequentially form the phagophore, autophagosome, amphisome, autolysosome and autophagic body.

    “Therefore, the use of TEM is a valid and important method both for the qualitative and quantitative analysis of changes in various autophagic structures that sequentially form the phagophore, autophagosome, amphisome, autolysosome and autophagic body (Fig. 1).43”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [43] source

      The Autophagic Pathways section describes the progression from phagophore to autophagosome, amphisome and autolysosome, and explains how electron microscopy identifies and classifies autophagic vacuoles. The Fusion Events section uses TEM images and a semiquantitative classification of early and late vacuoles. However, the paper cautions that morphology often cannot distinguish amphisomes from autolysosomes, and it does not establish autophagic bodies as a further sequential mammalian structure that TEM can separately analyze. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    It is possible to arrest autophagic flux at a given point and then record the time-dependent accumulation of an organelle, an organelle marker, a cargo marker or the entire cargo at the point of blockage.

    “Alternatively, it is possible to arrest the autophagic flux at a given point, and then record the time-dependent accumulation of an organelle, an organelle marker, a cargo marker or the entire cargo at the point of blockage; however, the latter assumes there is no feedback of the accumulating structure on its own rate of formation.23”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [23] source

      In the section “What is the advantage of a transient precursor in autophagosome biogenesis?” (page unavailable), the paper tracks labeled omegasomes and autophagosomes over time and discusses feedback. Our copy does not describe arresting autophagic flux at a blockage and measuring the resulting accumulation of organelles, markers, or cargo; searches for flux arrest and blockage found no such passage. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • The authors state this as an assumption: In methods that arrest autophagic flux at a given point and record time-dependent accumulation of a marker, there is no feedback of the accumulating structure on its own rate of formation.
  • May combine two claims. 156
  • Classified as probably not a substantial claim. 110

  • May combine two claims.

    Premisecitation

    This results in the preferential degradation of mitochondrial outer membrane proteins by the proteasome, while inner membrane proteins and mitochondrial DNA remain intact.

    “This results in the preferential degradation of mitochondrial outer membrane proteins by the proteasome, while inner membrane proteins and mitochondrial DNA 558 remain intact.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [558] source

      In Results, under “p62/SQSTM1 is not essential for mitochondrial degradation” (page unavailable), the paper reports that mitochondrial DNA decreased after CCCP treatment, contrary to the claim that it remained intact. It identifies autophagic and lysosomal degradation of depolarized mitochondria; it does not report preferential proteasomal degradation of outer-membrane proteins while inner-membrane proteins remain intact. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

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  • May combine two claims.

    Interpretationcitation

    Post-absorptive release of amino acids is strongly inhibited by infusion of insulin or exogenous supply of amino acids, suggesting it is mainly mediated by a lysosomal/autophagic mechanism of protein catabolism.

    “This post-absorptive release of amino acids is strongly inhibited by infusion of insulin or by exogenous supply of amino acids, suggesting that it is mainly mediated by a lysosomal/autophagic mechanism of protein catabolism.[643][644][645][646][647][648][649][650]”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [643] source
    • cites [644] source
    • cites [645] source

      The Results (p. 5) report that amino-acid infusion did not significantly change outward amino-acid transport or intracellular amino-acid appearance, an index of proteolysis. The Discussion (pp. 6–7) says protein breakdown remained unchanged; net balance shifted from release to uptake because synthesis increased. The paper does not attribute that shift to inhibited lysosomal/autophagic catabolism or establish an effect of insulin infusion. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    Proximal tubule-specific ATG5-deficient mice display a degeneration phenotype earlier than podocyte-specific ATG5-deficient mice, suggesting autophagy and hence LC3 levels might be more prominent in the former.

    “tubule-specific ATG5-deficient mice 656 display a degeneration phenotype earlier than podocyte-specific ATG5-deficient mice, 657 suggesting that autophagy, and hence LC3 levels, might actually be more prominent in the former.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [656] source

      The Results report early cellular changes in proximal tubule-specific Atg5-deficient mice, and the paper describes eventual tubular degeneration. They also report suppressed LC3-I-to-LC3-II conversion after Atg5 deletion. Our copy does not compare the timing of degeneration or LC3 levels with podocyte-specific Atg5-deficient mice, so it does not support those comparative claims. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [657] source

      The section “Glomerular podocytes exert high levels of autophagy under basal conditions” (page not available) reports more detectable autophagosomes in podocytes than in other kidney cells and faster LC3-II accumulation in podocytes than in the tubular cells compared. This is contrary to the suggestion that autophagy and LC3 are more prominent in tubules. The paper reports late degeneration in podocyte-specific Atg5-deficient mice, but does not establish an earlier phenotype in proximal tubule-specific Atg5-deficient mice. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In a limited number of cases, autophagy has been established as the cause of cell death, although opposite results have been reported using analogous experimental settings.

    “In a limited number of cases, autophagy has been established as the cause of cell death; 97,199,[658][659][660][661][662][663][664] although opposite results have been reported using analogous experimental settings.665”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [97] source
    • cites [199] source

      The citation is attached to the claim that autophagy causes cell death, not to the later clause about opposite results. The Discussion (page unavailable) instead says this work supports the hypothesis that autophagy is required to maintain cellular viability; it reports accelerated senescence in autophagy-gene mutants. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

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    • cites [658] source

  • May combine two claims.

    Premisecitation

    Plant root tips cultured in nutrient-sufficient medium display constitutive autophagic flux (i.e., a basal level), which is enhanced in nutrient-deprived medium.

    “Finally, plant root tips cultured in nutrient-sufficient medium display constitutive autophagic flux (i.e., a basal level), which is enhanced in nutrient-deprived medium.696,778,779”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [696] source

      In the Results section on barley root tips (page unavailable), pilot experiments found no difference in autolysosome accumulation with versus without sucrose, contrary to the claimed enhancement in nutrient-deprived medium. The Discussion speculates about normal low-level autophagic activity but does not establish constitutive flux in nutrient-sufficient root tips. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

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    • cites [778] source
    • cites [779] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Autophagy was first detected by TEM in the 1950s.

    “Autophagy was first detected by TEM in the 1950s (reviewed in ref. 8).”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The impact of autophagic flux on cell death and human pathologies demands accurate tools to measure not only the current flux of the system, but also its capacity and its response time when exposed to a defined insult.

    “The impact of autophagic flux on cell death and human pathologies therefore demands accurate tools to measure not only the current flux of the system, but also its capacity, 20 and its response time, when exposed to a defined insult.21”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [20] source
    • cites [21] source

  • May combine two claims.

    Findingcitation

    mCherry has enhanced photostability and intensity compared to mRFP, an order of magnitude higher and comparable to GFP, enabling acquisition of images at similar exposure settings as GFP, thus minimizing potential bias in interpretation.

    “a putative advantage of mCherry over mRFP is its enhanced photostability and intensity, which are an order of magnitude higher (and comparable to GFP), enabling acquisition of images at similar exposure settings as are used for GFP, thus minimizing potential bias in interpretation.182”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [182] source

      The Introduction discusses red fluorescent proteins generally and attributes brightness and photostability to DsRed-Express2, but does not compare mCherry with mRFP. Searches for mCherry, the claimed order-of-magnitude difference, and GFP-like imaging exposure settings found no passage addressing those claims. Our copy therefore cannot verify the attributed comparison or its proposed effect on interpretation. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Dendra2 is an improved version of the green-to-red photoswitchable fluorescent protein Dendra, derived from the octocoral Dendronephthya sp.

    “Dendra2 is an improved version of the green-to-red photoswitchable fluorescent protein Dendra, which is derived from the octocoral Dendronephthya sp.184”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [184] source

      The Methods sections on GFP–Hsp70 construction and confocal microscopy describe an EGFP fusion protein and LysoTracker staining, not Dendra2. Searches for Dendra2, Dendronephthya and photoconvertible coral proteins found no passage in our copy addressing Dendra2’s relationship to Dendra or its origin. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationreasoning

    It is possible to use anti-LC3/Atg8 antibodies for immunocytochemistry or immunohistochemistry, procedures that have the advantages of detecting the endogenous protein, obviating the need for transfection and/or the generation of a transgenic organism, as well as avoiding potential artifacts resulting from overexpression.

    “It is also possible to use anti-LC3/Atg8 antibodies for immunocytochemistry or immunohistochemistry, 140,[204][205][206][207][208][209] procedures that have the advantages of detecting the endogenous protein, obviating the need for transfection and or the generation of a transgenic organism, as well as avoiding potential artifacts resulting from overexpression”
    Quote in the paperNot a substantial claim3 of 7 sources checked
    • cites [140] source
    • cites [204] source
    • cites [205] source
    • cites [206] source
    • cites [207] source
    • cites [208] source
    • cites [209] source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The Venus variant of YFP is brighter than mRFP and less sensitive to pH than GFP.

    “Another alternative to mRFP or mCherry is to use the Venus variant of YFP, which is brighter than mRFP and less sensitive to pH than GFP.186”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [186] source

      The DNA constructions and LC3-II localization sections describe a GFP–LC3 construct and its use in microscopy. Searches for Venus/YFP, mRFP, brightness, and pH sensitivity found no passage comparing these fluorescent proteins, so our copy does not address the attributed claim. The indexed passages provide no page numbers. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    EGFP has a relatively neutral pH optimum for fluorescence, so its signal may diminish quickly during live cell imaging due to the acidic environment of the lysosome.

    “a caution concerning the use of the EGFP fluorescent protein for microscopy is that this fluorophore has a relatively neutral pH optimum for fluorescence, 180 so that its signal may diminish quickly during live cell imaging due to the acidic environment of the lysosome.”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Interpretationcitation

    The pH optimum of EGFP is important to consider when using GFP-LC3 constructs, as the original GFP-LC3 marker uses the EGFP variant, which may result in a reduced signal upon the formation of amphisomes or autolysosomes.

    “The pH optimum of EGFP is important to consider when using GFP-LC3 constructs, as the original GFP-LC3 marker 187 uses the EGFP variant, which may result in a reduced signal upon the formation of amphisomes or autolysosomes.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [187] source

      The DNA and siRNA Transfection section (page unavailable) identifies the construct used in this study as EGFP-LC3, supporting the claim that a GFP-LC3 construct uses EGFP. The paper does not describe it as the original GFP-LC3 marker or discuss EGFP’s pH optimum or reduced signal in amphisomes or autolysosomes. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Late/degradative autophagic vacuoles and autolysosomes (AVd) usually have only one limiting membrane, and contain cytoplasmic material and/or organelles at various stages of degradation.

    “Late/degradative autophagic vacuoles and autolysosomes (AVd) usually have only one limiting membrane, and contain cytoplasmic material and/or organelles at various stages of degradation (Fig. 2A and B).43,47”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [43] source

      The Autophagic Pathways section defines AVd as containing partially degraded cytoplasmic material and says AVd can be amphisomes or autolysosomes. The copy does not establish that AVd usually have only one limiting membrane; the Figure 2 description in Fusion Events with Endosomes and Lysosomes describes an AVd with both an inner and an outer membrane. Page numbers are not available in the indexed passages. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [47] source

      The section on leupeptin-induced alterations (page unavailable) reports that hepatocytic lysosomes contained autophagocytosed cytoplasm at various stages of degradation. The Discussion (page unavailable) identifies secondary lysosomes with autophagic contents as autolysosomes. Our copy does not state that these vacuoles usually have only one limiting membrane; it specifies double or multiple membranes for autophagosomes instead. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Oxidative stress inhibits MTOR, thus allowing autophagy despite concomitant activation of AKT1.

    “TORC1 is downstream of AKT1; however, oxidative stress inhibits MTOR, thus allowing autophagy, despite the concomitant activation of AKT1.115”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [115] source

      The Abstract (page unavailable) reports that increased oxidative stress after stable PINK1 silencing activates mitophagy, an autophagy-based process. Neither that passage nor the Discussion passages establish that oxidative stress inhibits MTOR, that TORC1 is downstream of AKT1, or that AKT1 is activated concurrently. (LLM, conf=0.78)

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  • May combine two claims.

    Premisecitation

    ATG16L1 has been used to monitor the movement of plasma membrane as a donor for autophagy, and thus an early step in the process; ATG16L1 is located on phagophores, but not on completed autophagosomes.

    “ATG16L1 has been used to monitor the movement of plasma membrane as a donor for autophagy, and thus an early step in the process; ATG16L1 is located on phagophores, but not on completed autophagosomes.269,411”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [269] source

      The Introduction discusses GFP-Atg8p and LC3 as autophagy markers, and the paper reports vacuolar turnover in yeast. Searches for ATG16L1, phagophores, and plasma membrane as an autophagy membrane donor found no passage addressing ATG16L1’s use or its location on developing versus completed autophagosomes. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [411] source

  • May combine two claims.

    Premisecitation

    ATG5, ATG12, and ATG16L1 associate with the phagophore and have been detected by fluorescence or immunofluorescence.

    “ATG5, ATG12, and ATG16L1, associate with the phagophore and have been detected by fluorescence or immunofluorescence (Fig. 16).400,401”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [400] source

      In “Apg16L localizes to autophagic isolation membrane” (page not provided), the paper reports that the Apg12–Apg5 conjugate associates with isolation membranes and shows fluorescently tagged Apg5 and Apg16L co-labeling those membranes. Fig. 8 also documents immunofluorescence detection of Apg16L. The paper does not show Apg12 individually detected there by fluorescence or immunofluorescence. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [401] source

      The Results sections “Apg5 Colocalizes with Punctate LC3 Structures” and “Apg5 Localizes to Isolation Membranes” report GFP-Apg5 on isolation membranes and anti-Apg12 staining overlapping its punctate signals. These observations support the ATG5 and ATG12 portion, but our copy does not report fluorescence or immunofluorescence detection of ATG16L1 at the phagophore. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In some cells ULK1 has functions in addition to autophagy, such as in axonal transport and outgrowth, and its activity state may reflect its role in these processes.

    “Furthermore, in some cells ULK1 has functions in addition to autophagy, such as in axonal transport and outgrowth, and its activity state may thus reflect its role in these processes.[390][391][392][393][394][395]”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [390] source
    • cites [391] source

      The Results section reports that Unc-51 has roles beyond autophagy, including a likely role in axonal transport; the Discussion also notes its requirement for axon outgrowth in worms and mice. The paper does not establish that ULK1’s activity state reflects those roles, so that part remains an inference rather than a finding of this source. No page numbers are available in our copy. (LLM, conf=0.82)

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    • cites [392] source

      The paper identifies ULK1 among kinases implicated in axonal events and in its screen of neurite outgrowth and retraction (opening summary; Discussion, page not available). It does not establish a role for ULK1 in axonal transport or test whether ULK1's activity state reflects a role in these processes. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    ATG14-GFP or BATS-GFP detected by fluorescence microscopy or TEM can be used as a phagophore marker; however, ATG14 is not localized exclusively to phagophores, as it can also be detected on mature autophagosomes as well as the ER.

    “ATG14-GFP or BATS-GFP detected by fluorescence microscopy or TEM can be used as a phagophore marker; however, ATG14 is not localized exclusively to phagophores, as it can also be detected on mature autophagosomes as well as the ER.409,410”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [409] source

      The Results (page unavailable) use GFP-tagged BATS in fluorescence microscopy and show that BATS puncta colocalize with Atg16, a phagophore marker. The Results also describe ATG14L association with the ER. They do not show ATG14 on mature autophagosomes or demonstrate GFP-tagged ATG14/BATS as a marker detected by TEM; TEM is used to examine autophagic vacuoles. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [410] source

      The Results and discussion (page unavailable) show GFP-Atg14L puncta partially colocalizing with Atg16L, a phagophore marker, and show Atg14L at the ER. They do not establish that Atg14L is present on mature autophagosomes: LC3 marks both phagophores and completed autophagosomes. The passages do not support BATS-GFP or TEM as a phagophore-marker method. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In Arabidopsis and possibly other eukaryotes, ATG1 and ATG13 proteins are targets of autophagy, so their levels may drop substantially under conditions that induce autophagic turnover.

    “Finally, in Arabidopsis and possibly other eukaryotes, the ATG1 and ATG13 proteins are targets of autophagy, which means that their levels may drop substantially under conditions that induce autophagic turnover.174”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [174] source

      The Introduction (page unavailable) describes the Atg1/Atg13 complex as a regulator of autophagy in budding yeast, not as a target of autophagic degradation. Searches for Arabidopsis and for declining ATG1/ATG13 protein levels found no passage addressing the claimed turnover. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In Arabidopsis and some mammalian cells, essentially all of the ATG5 and ATG12 proteins exist in the conjugated form and the expression levels do not change, at least during short-term starvation.

    “In Arabidopsis and some mammalian cells it appears that essentially all of the ATG5 and ATG12 proteins exist in the conjugated form and the expression levels do not change, at least during short-term starvation.150,[400][401][402]”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [150] source

      The paper’s description of the conjugation cascades says ATG12 attaches to ATG5, but gives no fraction of either protein present in that form. Its nutritional-stress results concern maize ATG transcripts and ATG8, not ATG5–ATG12 protein levels in Arabidopsis or mammalian cells during short-term starvation. Searches for the conjugated fraction and starvation-related changes did not locate a passage addressing the claim. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [400] source

      The section “Apg12-Apg5 and Apg16L form a ~800 kDa protein complex” (page not provided) says that almost all Apg5 in wild-type mouse ES cells was conjugated to Apg12. It does not establish that almost all Apg12 was conjugated, address that proportion in Arabidopsis, or report unchanged ATG5 and ATG12 expression during short-term starvation. Its statement that complex formation was unaffected by nutrient conditions concerns the complex, not protein expression levels. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [401] source

      The Results sections “Production of Apg5-deficient Cells” and “Apg5 Colocalizes with Punctate LC3 Structures” report that nearly all Apg5 in the mouse embryonic stem-cell transformants was conjugated with Apg12 and that the total amount of the conjugate did not change significantly after amino-acid starvation. The paper does not establish the same findings for Arabidopsis, nor does these passages show that essentially all Apg12 was conjugated or that the expression levels of both individual proteins were unchanged. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Yeast Atg14 is the autophagy-specific subunit of the Vps34 complex I, and a human homolog named ATG14 (ATG14L/BARKOR) has been identified.

    “Yeast Atg14 is the autophagy-specific subunit of the Vps34 complex I, 404 and a human homolog, named ATG14 (ATG14L/BARKOR), has been identified.[405][406][407][408]”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [404] source
    • cites [405] source
    • cites [406] source

      The passage describing protein identification (section labeled Abstract; page unavailable) reports a newly identified mammalian protein with modest similarity to yeast Atg14 and names it Atg14L. However, the protein was isolated from mouse tissues, and our copy does not identify it as a human homolog or use the name BARKOR. The yeast-complex point is also described in the paper, but the citation in question follows the human-homolog point. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    There are two isoforms of WIPI2 (2B and 2D), and in C. elegans WIPI4 (EPG-6) has been identified as the WIPI homolog required for autophagy.

    “It should be noted that there are two isoforms of WIPI2 (2B and 2D), 421 and in C. elegans WIPI4 (EPG-6) has been identified as the WIPI homolog required for autophagy.422”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [421] source

      Citation 422 is attached to the C. elegans clause. The paper’s Discussion addresses human WIPI-1 and WIPI-2, but the passages found do not identify WIPI4/EPG-6 or establish its requirement for autophagy in C. elegans (searched: C. elegans WIPI4 EPG-6; EPG-6 WIPI-4 Caenorhabditis elegans). The WIPI2 isoform clause has a separate citation, 421. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [422] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The WatershedCounting3D plug-in for ImageJ may be useful for obtaining an objective number of puncta per cell.

    “For this purpose, the WatershedCounting3D plug-in for ImageJ may be useful.211,212”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [211] source

      The passages available from our copy are lists of links with no section or page labels. Searches for WatershedCounting3D, ImageJ, and objective puncta counting found no passage addressing whether the plug-in is useful for obtaining a puncta count per cell. (LLM, conf=0.69)

      Experimental — accuracy not yet measured
    • cites [212] source

      The Fluorescence microscopy section describes scoring macrophages by their Atg7-labelled structures, but does not mention ImageJ or the WatershedCounting3D plug-in. Searches for the plug-in, ImageJ, and automated puncta counting found no passage addressing whether it is useful for obtaining an objective count per cell. Page numbers were not available in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ZFYVE1 partially colocalizes with WIPI1 upon nutrient starvation, and also with WIPI2.

    “ZFYVE1 partially colocalizes with WIPI1 upon nutrient starvation, 421 and also with WIPI2.418”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [421] source
    • cites [418] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    A population of BECN1 may localize in proximity to the trans-Golgi network, and it is also present at the ER and mitochondria.

    “Although a population of BECN1 may localize in proximity to the trans-Golgi network, 433 it is also present at the ER and mitochondria.425”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [433] source
    • cites [425] source

  • May combine two claims.

    Premisecitation

    Elimination of DRAM1 by siRNA blocks autophagy as effectively as elimination of BECN1, indicating it is an essential component for this process, although its mechanism of action is not known.

    “The elimination of DRAM1 by siRNA blocks autophagy, 439,440 as effectively as elimination of BECN1, indicating it is an essential component for this process, although its mechanism of action is not known.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [439] source

      The Results section reports that siRNA knockdown of DRAM and BECN1 each prevented UV-induced degradation of VRK1 in WS1 fibroblasts. The Discussion says DRAM’s role is not yet known. The paper does not show that DRAM knockdown blocks autophagy generally, or establish that it does so as effectively as BECN1 knockdown across the process. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [440] source

      In the section “Evidence for the interplay between JNK and p53-DRAM signalling pathways in the regulation of autophagy” (page unavailable), the paper says DRAM silencing or depletion prevents 2-methoxyestradiol-induced autophagy in Ewing sarcoma cells. It does not specify siRNA in the passages available, and that context-specific result does not establish that DRAM1 elimination blocks autophagy generally. The paper also leaves DRAM’s mechanism of action open. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Transcriptional upregulation of Drosophila Atg8a and Atg8b is observed in the fat body following induction of autophagy at the end of larval development; Atg5, Atg6, Atg8a and Atg18 are upregulated in the ovary of starved flies; and an increase in Drosophila Atg8b is observed in cultured Drosophila l(2)mbn cells following starvation.

    “Transcriptional upregulation of Drosophila Atg8a and Atg8b is also observed in the fat body following induction of autophagy at the end of larval development, 453 Atg5, Atg6, Atg8a and Atg18 are upregulated in the ovary of starved flies, 454 and an increase in Drosophila Atg8b is observed in cultured Drosophila l(2)mbn cells following starvation (Gorski S, personal communication).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [453] source

      Split into 3 atoms — 1 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [454] source

      Split into 3 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites Gorski S, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In C. elegans, the FOXA transcription factor PHA-4 regulates the expression of several autophagy-related genes.

    “and in C. elegans, the FOXA transcription factor PHA-4 regulates the expression of several autophagy-related genes.458”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Increased expression of Atg5 in vivo after optic nerve axotomy in mice and increased expression of Atg7, Becn1 and Lc3a during neurogenesis at different embryonic stages in the mouse olfactory bulb are also seen.

    “Increased expression of Atg5 in vivo after optic nerve axotomy in mice 446 and increased expression of Atg7, Becn1 and Lc3a during neurogenesis at different embryonic stages in the mouse olfactory bulb are also seen.447”
    Quote in the paperNot a substantial claim2 of 2 sources checked

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An autophagy-dedicated microarray was developed as a high-throughput tool to simultaneously monitor the transcriptional regulation of all genes involved in, and related to, autophagy.

    “and an autophagy-dedicated microarray was developed as a high-throughput tool to simultaneously monitor the transcriptional regulation of all genes involved in, and related to, autophagy.444”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    DRAM1 is detected as a subpopulation in the Golgi and cis-Golgi, colocalizing with GOLGB1/giantin and GOLGA2/GM130, and also in early and late endosomes and lysosomes, colocalizing with EEA1 and LAMP2.

    “It is detected as a subpopulation in the Golgi and cis-Golgi, colocalizing with GOLGB1/giantin and GOLGA2/ GM130, and also in early and late endosomes and lysosomes, colocalizing with EEA1 and LAMP2.439”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [439] source

      The Results section, “A subpopulation of DRAM colocalizes in Golgi apparatus and endosome vesicles with VRK1” (page unavailable), reports partial colocalization with giantin and colocalization with GM130, EEA1, and LAMP2. It identifies EEA1 as an early-endosome marker and LAMP2 as a lysosomal marker, but does not establish localization in late endosomes. Figure 3 describes these observations in cells transfected to express DRAM. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Autophagy is critical for Drosophila midgut cell death, which is accompanied by transcriptional upregulation of all of the Atg genes tested, including Atg8a.

    “Autophagy is critical for Drosophila midgut cell death, which is accompanied by transcriptional upregulation of all of the Atg genes tested, including Atg8a (Fig. 18).199,451”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [199] source

      The Results passages concern ATG8 and ATG4 expression and autophagy in Arabidopsis, including induction under nitrogen starvation, not Drosophila midgut cell death. Searches for Drosophila midgut cell death and Atg8a transcription found no passage addressing the claim in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [451] source

      The Abstract says that disrupting autophagy severely delays Drosophila larval midgut destruction and that several Atg genes are transcriptionally upregulated during midgut cell death. It does not say that all Atg genes tested were upregulated or identify Atg8a as one of them; it describes Atg8a as a marker of autophagic puncta. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Certain forms of macroautophagy are induced in a BECN1-independent manner, and are not blocked by PtdIns3K inhibitors.

    “It is important to be aware, however, that certain forms of macroautophagy are induced in a BECN1-independent manner, and are not blocked by PtdIns3K inhibitors.97,431”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [97] source

      The Results section (‘p62 bodies are degraded by autophagy’; page unavailable) reports that 3-methyladenine inhibits a step of autophagy and causes p62 accumulation. Our copy does not address whether any forms of macroautophagy are BECN1-independent or resist PtdIns3K inhibitors (searched: BECN1-independent, PtdIns3K inhibitors, 3-methyladenine). The p62 finding does not rule out the existence of other forms. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites [431] source

  • May combine two claims.

    Premisecitation

    Caspase-mediated cleavage of BECN1 inactivates BECN1-induced autophagy and enhances apoptosis in several cell types.

    “Interestingly, caspase-mediated cleavage of BECN1 inactivates BECN1-induced autophagy and enhances apoptosis in several cell types, 432”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [432] source

      The ‘Crosstalk between apoptosis and autophagy by Beclin 1’ section says caspase cleavage destroys Beclin 1’s pro-autophagic activity and produces fragments that sensitize cells to apoptotic signals. It gives HeLa cells as one example, but does not establish the claimed effect across several cell types. Our copy provides no page number for this section. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Autophagy is induced by the release of BECN1 from BCL2 by pro-apoptotic BH3 proteins, phosphorylation of BECN1 by DAPK at Thr119, or phosphorylation of BCL2 by MAPK8/JNK1 at Thr69, Ser70 and Ser87.

    “Autophagy is induced by the release of BECN1 from BCL2 by pro-apoptotic BH3 proteins, phosphorylation of BECN1 by DAPK (at Thr119, located in the BH3 domain), 426 or phosphorylation of BCL2 by MAPK8/JNK1 (at Thr69, Ser70 and Ser87).427,428”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [426] source
    • cites [427] source
    • cites [428] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    DRAM1 is a gene induced by activated TP53/p53 in response to different types of cellular stress, including DNA damage.

    “DRAM1 (damage-regulated autophagic modulator 1) is a gene induced by activated TP53/p53 in response to different types of cellular stress, including DNA damage.438,439”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [438] source
    • cites [439] source

      The Results section, “DRAM gene expression is induced in response to DNA damage” (page not provided), reports that several DNA-damaging agents activated DRAM expression alongside p53 accumulation, and that wild-type p53 induced DRAM expression. The copy does not establish DRAM induction in response to other types of cellular stress; its reference to several types of stress concerns autophagy more generally. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Detection of DRAM1 RNA is very easy by qRT-PCR during autophagy; however, detection of the DRAM1 protein is very difficult because of its small size and hydrophobicity.

    “Detection of DRAM1 RNA is very easy by qRT-PCR during autophagy; 438,439 however, detection of the DRAM1 protein is very difficult because of its small size and hydrophobicity, which makes generation of specific antibodies a complicated process, and in general these have very low sensitivity.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [438] source

      The citation is attached to the RNA-detection clause. The Results report measuring DRAM mRNA by real-time quantitative RT-PCR after p53 induction, but do not establish that detection is “very easy” or specifically describe qRT-PCR detection during autophagy. The later protein-detection clause follows the citation and is not assessed here. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [439] source

      The citation follows the RNA-detection clause. The qRT-PCR Methods describe detecting human DRAM RNA, and the Results report measuring DRAM gene expression by qRT-PCR. The paper does not characterize that detection as “very easy”; the protein-detection clause comes after the citation. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several studies implicate microRNAs in controlling the expression of proteins associated with autophagy.

    “Although less work has been done on post-transcriptional regulation, several studies implicate microRNAs in controlling the expression of proteins associated with autophagy.164,[468][469][470]”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [164] source
    • cites [468] source
    • cites [469] source

  • May combine two claims.

    Premisecitation

    Even increases in LC3 mRNA can be quite modest and are cell type- and organism-dependent.

    “Even increases in LC3 mRNA can be quite modest and are cell type-and organism-dependent.471”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [471] source

      The ‘LC3 as a Potential Biomarker for Autophagy’ passage in Detection of Starvation-induced Autophagy reports weak LC3 mRNA upregulation—up to threefold—in starved HepG2 cells. It does not establish that LC3 mRNA increases vary by cell type or organism; the other cell and mouse observations concern LC3 protein rather than mRNA. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Yeast pexophagy utilizes the processing of Pex14-GFP and Pot1 (thiolase)-GFP.

    “For example, yeast pexophagy utilizes the processing of Pex14-GFP and Pot1 (thiolase)-GFP, 494,495”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [494] source
    • cites [495] source

      The Results section (‘The MAPK Slt2p is required for pexophagy’; page unavailable) describes monitoring yeast pexophagy through cleavage of Pot1p-GFP, identifying Pot1p as thiolase. Our copy does not describe processing Pex14-GFP, so it supports only the Pot1-GFP part of the claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Yeast mitophagy requires both the Slt2 and Hog1 signaling pathways; activation and phosphorylation of Slt2 and Hog1 can be monitored with commercially available phospho-specific antibodies.

    “In addition, yeast mitophagy requires both the Slt2 and Hog1 signaling pathways; the activation and phosphorylation of Slt2 and Hog1 can be monitored with commercially available phospho-specific antibodies (Fig. 19).381”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CEBPB/C/EBPβ is a transcription factor that regulates autophagy in response to the circadian cycle.

    “Finally, CEBPB/C/EBPβ is a transcription factor that regulates autophagy in response to the circadian cycle.467”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    VMP1 is an essential autophagy gene that is conserved from Dictyostelium to mammals, and the VMP1 protein regulates early steps of the autophagic pathway.

    “VMP1 is an essential autophagy gene that is conserved from Dictyostelium to mammals, 232,462 and the VMP1 protein regulates early steps of the autophagic pathway.420”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [232] source

      The passages labeled Abstract discuss p62/SQSTM1 and its possible role in autophagy, not VMP1. Searches for VMP1, its conservation from Dictyostelium to mammals, and its role in early autophagy steps found no passage addressing the claim; the available passages provide no page numbers. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [462] source
    • cites [420] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A gene regulatory network named CLEAR (coordinated lysosomal enhancement and regulation) that controls both lysosome and autophagosome biogenesis was identified using a systems-biology approach.

    “A gene regulatory network, named CLEAR (coordinated lysosomal enhancement and regulation) that controls both lysosome and autophagosome biogenesis was identified using a systemsbiology approach.[463][464][465]”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [463] source

      The passage discussing CLEAR (section labeled Abstract; page unavailable) calls it the “Coordinated Lysosomal Expression and Regulation” network, not “enhancement and regulation.” The paper reports that TFEB co-regulates lysosome and autophagosome biogenesis, but it treats CLEAR as an existing network and does not say it identified the network using a systems-biology approach. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [464] source
    • cites [465] source

  • May combine two claims.

    Premisecitation

    TFEB activity is regulated by starvation, and can thus serve as a new tool for monitoring transcriptional regulation connected with autophagy.

    “TFEB activity is regulated by starvation, 463 and can thus serve as a new tool for monitoring transcriptional regulation connected with autophagy.”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    The relative amount of free GFP and the complete fusion protein is the relevant parameter for quantification; although it may not be possible to detect clear changes in the amount of the full-length chimera, especially under conditions of limited flux.

    “In this case the relative amount of the free GFP and the complete fusion protein is the relevant parameter for quantification; although it may not be possible to detect clear changes in the amount of the full-length chimera, especially under conditions of limited flux.29,36”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [29] source

      Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [36] source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The erythroid transcription factor GATA1 and its coregulator Friend of GATA1 induce the transcription of multiple genes encoding autophagy components; this developmentally regulated transcriptional response is coupled to increases in autophagosome number as well as the percent of cells that contain autophagosomes.

    “Along these lines, the erythroid transcription factor GATA1 and its coregulator Friend of GATA1 induce the transcription of multiple genes encoding autophagy components; this developmentally regulated transcriptional response is coupled to increases in autophagosome number as well as the percent of cells that contain autophagosomes.466”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    In cells, it is also possible to measure the release of an unlabeled amino acid by chromatographic methods, thereby obviating the need for prelabeling; however, amino acid release is also regulated by protein synthesis.

    “In cells, it is also possible to measure the release of an unlabeled amino acid by chromatographic methods, thereby obviating the need for prelabeling; 490 however, it is important to keep in mind that amino acid release is also regulated by protein synthesis, which in turn is modulated by many different factors.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [490] source

      The available indexed passages are an IRIS catalog entry (labeled Abstract, no page number), not the paper’s substantive text. Searches for chromatographic measurement of unlabeled amino-acid release and for regulation of release by protein synthesis found no passage addressing either point, so the claim cannot be verified from our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    GFP-Vid24, Vid30-GFP and Sec28-GFP colocalize with actin during prolonged glucose starvation and for up to 30 min following glucose replenishment in wild-type cells; however, colocalization is less obvious by the 60 min time point.

    “GFP-Vid24, Vid30-GFP and Sec28-GFP colocalize with actin during prolonged glucose starvation and for up to 30 min following glucose replenishment in wild-type cells; however, colocalization is less obvious by the 60 min time point.540,543”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [540] source
    • cites [543] source

  • May combine two claims.

    Premisecitation

    Mutants such as vac8D and tlg2D accumulate prApe1 under rich conditions, but not during autophagy.

    “In addition, mutants such as vac8D and tlg2D accumulate prApe1 under rich conditions, but not during autophagy.380,532”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [380] source

      The section “Vac8p Is Not Essential for Autophagy” reports negligible prAPI delivery in vac8Δ cells in nitrogen-containing medium and near-wild-type delivery during starvation. The paper names tlg2 among mutants primarily defective in one pathway or the other, but the passages do not establish the claimed rich-medium versus autophagy pattern for tlg2Δ. Page numbers are unavailable in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [532] source

      The sections “API maturation is the major biosynthetic trafficking defect in tlg2∆ cells” and “Rapamycin-induced autophagy bypasses the API transport block in tlg2∆ cells” report blocked API maturation in tlg2∆ cells under ordinary conditions and rapid, near-complete proAPI maturation when autophagy is induced. The cited paper does not report the corresponding result for vac8∆ mutants, so it supports only the tlg2∆ example. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Vid vesicles can be purified by fractionation and gradient centrifugation; western blotting analysis using antibodies against organelle markers and Fbp1, and the subsequent verification of fractions by EM facilitate their identification.

    “Vid vesicles can be purified by fractionation and gradient centrifugation; western blotting analysis using antibodies against organelle markers and Fbp1, and the subsequent verification of fractions by EM facilitate their identification.539”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Interpretationcitation

    Immunodetection of LC3 as definite puncta is possible in paraffin-embedded tissue sections and fresh frozen tissue by immunohistochemistry or immunofluorescence; however, this methodology has not received extensive evaluation and does not lend itself well to dynamic assays.

    “Immunodetection of LC3 as definite puncta is possible in paraffin-embedded tissue sections and fresh frozen tissue, by either immunohistochemistry or immunofluorescence; 140,[634][635][636][637] however, this methodology has not received extensive evaluation, and does not lend itself well to dynamic assays.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [140] source
    • cites [634] source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [635] source

  • May combine two claims.

    Premisecitation

    Multiple disease states have been associated with altered activity of the ubiquitin-proteasome system, complicating use of amino acid exchange rate as a marker of autophagy.

    “In addition, multiple disease states have been associated with an altered activity of the ubiquitin-proteasome system, further complicating the use of the amino acid exchange rate as a marker of autophagy.654”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [654] source

      The Main section links UPS malfunction to several disease categories, including cancer, cardiovascular disease and neurodegenerative disorders. It does not discuss amino acid exchange rate as a marker of autophagy or say that altered UPS activity complicates its use. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    PCP allows proteome-wide recording of protein gradient profiles.

    “Compared with classical biochemical approaches, PCP allows the proteome-wide recording of protein gradient profiles.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [278] source

      The Results section (page unavailable) describes density-gradient fractionation and mass-spectrometric measurement of protein distributions across fractions. It does not identify the method as PCP or establish the claimed proteome-wide scope or comparison with classical biochemical approaches. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    Twelve h of fasting in mice may be sufficient to trigger autophagy in muscle, but the appropriate time should be determined empirically.

    “Twelve h of fasting in mice may be sufficient to trigger autophagy in muscle, 639,640 but the appropriate time should be determined empirically.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [639] source
    • cites [640] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Bone marrow transfer has been used to document in vivo the role of autophagy in the reverse cholesterol transport pathway from peripheral tissues or cells to the liver for secretion in bile and for excretion.

    “Bone marrow transfer has been used to document in vivo the role of autophagy in the reverse cholesterol transport pathway from peripheral tissues or cells (e.g., macrophages) to the liver for secretion in bile and for excretion, 638”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [638] source

      The Results section, “Lipophagy Contributes to RCT In Vivo” (page unavailable), reports that autophagy-deficient macrophages showed reduced cholesterol transport to plasma, liver, gallbladder, and feces. But the Experimental Procedures, “In Vivo RCT Studies” (page unavailable), describe injecting labeled macrophages into mice, not bone marrow transfer. The in vivo role is supported; the named method is not. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Chloroquine suppresses the immunological response in a manner not due to its pH-dependent lysosomotropic accumulation and through its pH-dependent inhibition of antigen presentation.

    “Chloroquine suppresses the immunological response in a manner that is not due to its pHdependent lysosomotropic accumulation (chloroquine interferes with lipopolysaccharide-induced TNF/TNFa gene expression by a nonlysosmotropic mechanism), 651 as well as through its pHdependent inhibition of antigen presentation.652”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [651] source
    • cites [652] source

  • May combine two claims.

    Interpretationcitation

    Keima is a quantitative technique capable of monitoring fusion of autophagosomes with lysosomes (formation of an autolysosome) and does not depend on analysis of LC3.

    “This quantitative technique is capable of monitoring the fusion of autophagosomes with lysosomes, that is, the formation of an autolysosome, and the assay does not depend on the analysis of LC3.”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Interpretationcitation

    Cellular viability is related to high CTSB activity and low CTSD/cathepsin D activities; therefore, the appearance of opposite levels may indicate initiation of autophagy (lysosome)-dependent cell death.

    “For example, cellular viability is related to high CTSB activity and low CTSD/ cathepsin D activities.642 Therefore, the appearance of the opposite levels of activities may be one indication of the initiation of autophagy (lysosome)-dependent cell death.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [642] source

      In serum-deprived PC12 cells, the paper reports that cathepsin B activity falls while cathepsin D activity rises during cell death (III-1, p. 6), and describes a cathepsin-regulated death pathway downstream of autophagy (III-2, p. 10). But it does not establish high B and low D as a general condition for cellular viability: PC12 cells overexpressing B had improved survival even though their cathepsin D protein amount increased (Fig. 7, p. 10). (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    Cleavage of GFP-LC3 to generate free GFP can be evaluated, and this has been successfully performed in mouse liver, suggesting the GFP-LC3 cleavage assay may also be applied to in vivo studies.

    “In addition, cleavage of GFP-LC3 to generate free GFP can be evaluated.This has been successfully performed in mouse liver, 172,631 suggesting the GFP-LC3 cleavage assay may also be applied to in vivo studies.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [172] source
    • cites [631] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Adenoviral-mediated expression of the transcription factor CEBPB/C/EBPβ induces autophagy in hepatocytes.

    “Similarly, adenoviral-mediated expression of the transcription factor CEBPB/C/EBPβ induces autophagy in hepatocytes.467”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    The initial characterization of autophagy was due in large part to the inducing effects of glucagon, which appears to act through indirect inhibition of MTOR via the activation of STK11/LKB1-AMPK.

    “There are fewer compounds that act as inducers of autophagy, but the initial characterization of this process was due in large part to the inducing effects of glucagon, which appears to act through indirect inhibition of MTOR via the activation of STK11/LKB1-AMPK.615,616,761”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [615] source

      The Introduction (p. C11) calls glucagon a powerful inducer of autophagy in liver, and the results (p. C12) report numerous autophagic vacuoles in glucagon-injected rats compared with controls. This supports glucagon’s role in the early study of autophagy, but the paper does not address MTOR inhibition or STK11/LKB1–AMPK activation. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [616] source

      The Introduction (p. 437) says the discovery that glucagon induces autophagic vacuoles opened the way to studying autophagy under reproducible, timed conditions. The paper does not address the proposed STK11/LKB1–AMPK or MTOR mechanism, so it supports the historical and inducing-effects portion but not that mechanistic explanation. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [761] source

      The Results and Discussion report that glucagon represses mTOR signaling in perfused rat liver and is associated with phosphorylation of LKB1 and AMPK, supporting the proposed indirect pathway. Our copy does not address glucagon-induced autophagy or the claim that glucagon was important to autophagy’s initial characterization. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Overexpression or post-translational activation of the gene encoding TFEB induces autophagy; TFEB is a transcriptional regulator of the biogenesis of both lysosomes and autophagosomes.

    “For example, this can be achieved either through overexpression or post-translational activation of the gene encoding TFEB (see Transcriptional and translational regulation), a transcriptional regulator of the biogenesis of both lysosomes and autophagosomes.463,464”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [463] source
    • cites [464] source

  • May combine two claims.

    Premisecitation

    ICP34.5, viral BCL2 homologs and viral CFLAR/FLIP of herpesviruses block autophagosome formation.

    “Along these lines ICP34.5, viral BCL2 homologs and viral CFLAR/FLIP of herpesviruses block autophagosome formation, 425,580,759”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [425] source
    • cites [580] source
    • cites [759] source

  • May combine two claims.

    Premisecitation

    MTOR is a major regulatory protein that is part of the insulin signaling pathway and controls processes other than autophagy, so rapamycin will ultimately affect many metabolic pathways.

    “MTOR is a major regulatory protein that is part of the insulin signaling pathway, and it controls processes other than autophagy, so that rapamycin will ultimately affect many metabolic pathways.379,[762][763][764]”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [379] source

      The Introduction (page not provided) describes Tor as a nutrient-responsive regulator and rapamycin as its inhibitor; it also reports effects beyond autophagy, including cell-cycle arrest and glycogen accumulation. The Discussion identifies mammalian Tor (mTOR), but our copy does not place it in the insulin signaling pathway or establish that rapamycin affects many metabolic pathways in mammals. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [762][763][764]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    Claim (b) is stronger than claim (a) and needs proof that inhibiting autophagy prevents cell death.

    “Clearly claim (b) is stronger than claim (a), and needs to be justified by proof that inhibiting autophagy, through either genetic or chemical means, prevents cell death.669”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    A substantial amount of neuronal cell death occurs during neurogenesis, making it problematic to carry out a correct analysis in vivo or ex vivo.

    “however, a substantial amount of neuronal cell death occurs during neurogenesis, making it problematic to carry out a correct analysis in vivo or ex vivo.677,678”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [677] source
    • cites [678] source

      The indexed passages are repository file listings, not passages from the paper; they have no section or page numbers. Searches for neuronal death during neurogenesis and for difficulties analyzing it in vivo or ex vivo returned no substantive text, so the claim cannot be checked against our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Activation of the lysosomal two pore channel (TPC) by nicotinic acid adenine dinucleotide phosphate (NAADP) induces autophagy, which can selectively be inhibited by the TPC blocker NED-19, or by pre-incubation with BAPTA, showing that lysosomal calcium also modulates autophagy.

    “The activation of the lysosomal two pore channel (TPC), by nicotinic acid adenine dinucleotide phosphate (NAADP) induces autophagy, which can selectively be inhibited by the TPC blocker NED-19, or by pre-incubation with BAPTA, showing that lysosomal calcium also modulates autophagy.244”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [244] source

      Split into 2 atoms — 0 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 2 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Autophagy may also be regulated by the release of calcium from the endoplasmic reticulum under stress conditions.

    “Autophagy may also be regulated by the release of calcium from the endoplasmic reticulum under stress conditions; 212,726,766”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [212] source

      The Introduction links autophagy to cellular stress and discusses several regulators, but does not mention calcium release. The section on secretory traffic discusses vesicles from the endoplasmic reticulum, not calcium signaling. Searches for calcium release and ER-stress regulation found no passage addressing the claimed mechanism. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [726] source
    • cites [766] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In vivo treatment of embryos with cadmium results in an increase in autophagy, probably to counter the stress, allowing cell survival through the elimination/recycling of damaged structures.

    “In vivo treatment of embryos with cadmium results in an increase in autophagy, probably to counter the stress, allowing cell survival through the elimination/ recycling of damaged structures.632”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    Some PAMP recognition pathways, like TLR-mediated RNA recognition, can influence autophagy by either masking any inhibitory effect or compromising autophagy independent of the knockdown probe.

    “Some of these, like TLR-mediated RNA recognition, 756 can influence autophagy by either masking any inhibitory effect or compromising autophagy independent of the knockdown probe.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [756] source

      The Introduction and the section ‘ssRNA induces autophagy through TLR7’ show that RNA recognition through TLR7 can induce autophagy; the latter also notes that experimental manipulations sometimes increased background LC3 puncta. The paper does not establish that this response masks a knockdown probe’s inhibitory effect or compromises autophagy independently of the probe. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Upon starvation-induced multicellular development in Dictyostelium, autophagy (or at least Atg1) is required to protect against starvation-induced cell death, allowing vacuolar developmental cell death to take place instead.

    “For example, upon induction by starvation of multicellular development in the protist Dictyostelium, autophagy (or at least Atg1) is required to protect against starvation-induced cell death, allowing vacuolar developmental cell death to take place instead.672,673”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [672] source
    • cites [673] source

  • May combine two claims.

    Premisecitation

    Some immune cell types such as dendritic cells seem to tolerate loss of autophagy fairly well, whereas others such as T and B cells are compromised in their development and function after autophagy inhibition.

    “In this respect it is noteworthy that some cell-types of the immune system such as dendritic cells 260 seem to tolerate loss of autophagy fairly well, whereas others such as T and B cells are compromised in their development and function after autophagy inhibition.757,758”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [260] source
    • cites [757] source

      The Abstract and Results and Discussion (pages not provided) report reduced peripheral T- and B-cell numbers in Atg5-deficient chimeras and impaired T-cell proliferation, supporting developmental and functional compromise in lymphocytes. The paper does not show impaired B-cell function; it also reports that Atg5-deficient T cells underwent full maturation. The dendritic-cell comparison has a separate citation in the citing sentence and is not attributed to this paper. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [758] source

      The Introduction and the section on peripheral B-1 cells report impaired B-cell development and reduced B-1a cell numbers after ATG5 loss. The Introduction also reports a cited finding on T-cell survival and proliferation. This paper does not establish impaired immune-cell function generally or address whether dendritic cells tolerate autophagy loss; the citing sentence assigns those points to other citations. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    M2 of influenza virus and HIV Nef block autophagosome degradation.

    “M2 of influenza virus and HIV Nef block autophagosome degradation.278,760”
    Quote in the paperNot a substantial claim2 of 2 sources checked

  • May combine two claims.

    Premisecitation

    This stabilization results in the induction of BNIP3 and BNIP3L/NIX and 'hypoxia'-induced autophagy, even though the conditions are theoretically normoxic.

    “This results in the induction of BNIP3 and BNIP3L/NIX and”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    In chicken lymphoma DT40 cells and their knockout variant for all three inositol 1,4,5-trisphosphate receptor isoforms, no differences in basal levels of LC3-II can be observed up to 4 h after addition of fresh medium, but differences can be observed after longer times.

    “One example is the chicken lymphoma DT40 cells (see Chicken B-lymphoid DT40 cells below) and their knockout variant for all three inositol 1,4,5-trisphosphate receptor isoforms.241,772,773 In these cells, no differences in basal levels of LC3-II can be observed up to 4 h after addition of fresh medium, but differences can be observed after longer times (Vicencio JM, Szabadkai G, personal comm”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [241] source

      Split into 2 atoms — 0 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 2 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [772] source
    • cites [773] source
    • cites Vicencio JM, Szabadkai G, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The band running just below LC3-I at approximately 17.5 kDa may be a processing intermediate of LC3-I.

    “Note that the band running just below LC3-I at approximately 17.5 kDa may be a processing intermediate of LC3-I”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    An equivalent enzyme assay has not been developed for higher eukaryotic cells.

    “note that an equivalent assay has not been developed for higher eukaryotic cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Dendra2 has properties including a monomeric state, low phototoxic activation and efficient chromophore maturation, which make it suitable for real-time tracking of LC3 and SQSTM1.

    “This modified version of the fluorophore has certain properties including a monomeric state, low phototoxic activation and efficient chromophore maturation, which make it suitable for real-time tracking of LC3 and SQSTM1 (Fig. 8; Kaarniranta K, personal communication).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kaarniranta K, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    The band running just below LC3-I at approximately 17.5 kDa is detectable in freshly prepared homogenates but less visible after freeze-thaw.

    “it is detectable in freshly prepared homogenates, but is less visible after the sample is subjected to a freeze-thaw cycle.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    It is not always possible to detect endogenous Atg8/LC3.

    “but we note that it is not always possible to detect endogenous Atg8/LC3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    High levels of overexpressed GFP-LC3 can result in its nuclear localization, although the protein can still relocate to the cytosol upon starvation.

    “for example, high levels of overexpressed GFP-LC3 can result in its nuclear localization, although the protein can still relocate to the cytosol upon starvation”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning
    § Guidelines for the use and interpretation of assays for monitoring autophagy

    A block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity.

    “thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    In nonselective autophagy, the content of the autophagosome is morphologically identical to the cytoplasm, containing similar densities of ribosomes, and intact sequestered organelles are clearly identifiable.

    “In contrast, during nonselective autophagy, the content of the autophagosome is morphologically identical to the cytoplasm, containing similar densities of ribosomes, and intact sequestered organelles are clearly identifiable.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    In selective autophagy, the cargo should correspond to the specific substrate being targeted for sequestration, and bulk cytoplasm is essentially excluded.

    “In the case of selective autophagy, the cargo should correspond to the specific substrate being targeted for sequestration-bulk cytoplasm is essentially excluded.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisereasoning

    Autophagic activity includes not just increased synthesis or lipidation of Atg8/LC3 or an increase in formation of autophagosomes, but most importantly flux through the entire system, including lysosomes or the vacuole and subsequent release of breakdown products.

    “That is, autophagic activity includes not just the increased synthesis or lipidation of Atg8/LC3 (LC3 is a mammalian homolog of yeast Atg8), or an increase in the formation of autophagosomes, but, most importantly, flux through the entire 460”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    Autophagy is a highly dynamic, multi-step process.

    “One critical point is that autophagy is a highly dynamic, multi-step process.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    The focal degradation of cytoplasmic areas sequestered by the phagophore, which matures into the autophagosome, is the morphological hallmark of autophagy.

    “The focal degradation of cytoplasmic areas sequestered by the phagophore, which matures into the autophagosome, is the morphological hallmark of autophagy.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Increases in the level of PE-modified Atg8/LC3 (Atg8-PE/LC3-II), or even the appearance of autophagosomes, are not measures of autophagic flux per se, but can reflect the induction of autophagic sequestration and/or inhibition of autophagosome or amphisome clearance.

    “Thus, increases in the level of phosphatidylethanolamine (PE)-modified Atg8/LC3 (Atg8-PE/LC3-II), or even the appearance of autophagosomes are not measures of autophagic flux per se, but can reflect the induction of autophagic sequestration and/or inhibition of autophagosome or amphisome clearance.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Inefficient fusion with endosomes and/or lysosomes, or perturbation of the transport machinery, would inhibit autophagosome maturation to amphisomes or autolysosomes.

    “For example, inefficient fusion with endosomes and/or lysosomes, respectively, or perturbation of the transport machinery, 18 would inhibit autophagosome maturation to amphisomes or autolysosomes”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    It should not be assumed that an autophagic system is at steady-state in the strict biochemical meaning of this term.

    “It should not be assumed that an autophagic system is at steady-state in the strict biochemical meaning of this term, as this implies that the level of autophagosomes does not change with time, and the flux through the system is constant.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The use of autophagy markers such as LC3-II needs to be complemented by assays to estimate overall autophagic flux to permit a correct interpretation of the results.

    “Accordingly, the use of autophagy markers such as LC3-II needs to be complemented by assays to estimate overall autophagic flux, or flow, to permit a correct interpretation of the results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Premisereasoning
    § Guidelines for the use and interpretation of assays for monitoring autophagy

    No individual assay is guaranteed to be the most appropriate one in every situation.

    “we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    An accumulation of autophagosomes measured by TEM, GFP-LC3 dots, or LC3 lipidation could reflect induction of autophagy, reduction in autophagosome turnover, or inability of turnover to keep pace with increased autophagosome formation.

    “An accumulation of autophagosomes [measured by transmission electron microscopy (TEM) image analysis, as fluorescent GFP-MAP1LC3 (GFP-LC3) dots, or as LC3 lipidation on a western blot], could, for example, reflect induction of autophagy, reduction in autophagosome turnover, [14][15][16] or the inability of turnover to keep pace with increased autophagosome formation (Fig. 1).17”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [14][15][16]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [17] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Autophagy can be modulated at several steps, both positively and negatively.

    “Like other cellular pathways, it can be modulated at several steps, both positively and negatively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Cells tune autophagic flux to meet intracellular metabolic demands.

    “By responding to perturbations in the extracellular environment, cells tune autophagic flux to meet intracellular metabolic demands.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    Autophagic substrates need to be monitored dynamically over time to verify that they have reached the lysosome/vacuole and, when appropriate, are degraded.

    “Therefore, autophagic substrates need to be monitored dynamically over time to verify that they have reached the lysosome/vacuole, and, when appropriate, are degraded.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductnone given

    The GFP-Atg8 processing assay is not always performed in a quantitative manner; western blot exposures need to be in the linear range.

    “this assay is not always performed in a quantitative manner (for example, western blot exposures need to be in the linear range).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Study design and conductnone given

    Appropriate caution must be used with any enzyme assay regarding substrate concentrations and linearity.

    “appropriate caution must be used as with any enzyme assay regarding, for example, substrate concentrations and linearity”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    All tagged proteins are prone to show enrichment in lysosomes during nonspecific autophagy of the cytoplasm, especially at higher expression levels.

    “all tagged proteins are prone to show enrichment in lysosomes during nonspecific autophagy of the cytoplasm, especially at higher expression levels.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The lower pH of lysosomes relative to that of the vacuole may contribute to differences in detecting free GFP.

    “Alternatively, the lower pH of lysosomes relative to that of the vacuole may contribute to differences in detecting free GFP.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    GFP is relatively resistant to denaturation, and boiling for 5 min may be needed to prevent the folded protein from being trapped in the stacking gel during SDS-PAGE.

    “Also, GFP is relatively resistant to denaturation, and boiling for 5 min may be needed to prevent the folded protein from being trapped in the stacking gel during SDS-PAGE.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Dendra2 is capable of irreversible photoconversion from a green to a red fluorescent form, but can be used also as a normal GFP or RFP vector.

    “Dendra2 is capable of irreversible photoconversion from a green to a red fluorescent form, but can be used also as a normal GFP or RFP vector.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    The nature of multi-lamellar membrane structures observed in autophagy-inducing conditions is not fully understood.

    “The nature of these structures is not fully understood.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    It is better to use a saturating concentration of lysosomal inhibitors when performing an autophagic flux assay.

    “it would be better to use a saturating concentration of lysosomal inhibitors when performing an autophagic flux assay.172”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [172] source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Lysosomotropic compounds stabilize GFP by neutralizing the pH in the lysosome.

    “although lysosomotropic compounds should help stabilize GFP by neutralizing the pH in the lysosome”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    The cytoplasmic origin of the contents must still be morphologically identifiable if morphology is the only criterion being used for identification of autolysosomes.

    “The cytoplasmic origin of the contents must still be morphologically identifiable, if morphology is the only criterion that is being used for the identification of autolysosomes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    In quickly frozen and fractured cells, the fracture runs preferentially along the hydrophobic plane of the membranes, allowing characterization of the limiting membranes of different types of autophagic vacuoles and visualization of their limited protein intramembrane particles.

    “In quickly frozen and fractured cells the fracture runs preferentially along the hydrophobic plane of the membranes, allowing characterization of the limiting membranes of the different types of autophagic vacuoles and visualization of their limited protein intramembrane particles (IMPs, or integral membrane proteins).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Lysosomotropic compounds do not have an immediate effect on lysosomal hydrolase activity.

    “they do not have an immediate effect on lysosomal hydrolase activity”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    GFP is more sensitive to mammalian lysosomal hydrolases than to the degradative milieu of the yeast vacuole.

    “Apparently, GFP is more sensitive to mammalian lysosomal hydrolases than to the degradative milieu of the yeast vacuole.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    The GFP-Atg8/LC3 processing assay is a convenient way to monitor autophagy, but it does not work in all cell types, and is not as easy to quantify as enzyme-based assays.

    “The GFP-Atg8/LC3 processing assay (the generation of free GFP within the vacuole/lysosome) is a convenient way to monitor autophagy, but it does not work in all cell types, and is not as easy to quantify as enzyme-based assays.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    It is critical that any chimeras be constructed with the fluorescent tag at the N terminus of Atg8/LC3.

    “Accordingly, it is critical that any chimeras be constructed with the fluorescent tag at the N terminus of Atg8/LC3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Assays for Atg1/ULK1 can provide detailed insight into the induction of autophagy, but are not a direct measurement of the process.

    “Conclusion: Assays for Atg1/ULK1 can provide detailed insight into the induction of autophagy, but are not a direct measurement of the process.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Monitoring the endogenous protein depends on the ability to detect it in the system of interest.

    “Monitoring the endogenous protein, however, obviously depends on the ability to detect it in the system of interest.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Endogenous WIPI1 puncta cannot be detected in many cell types, and the level of transiently expressed GFP-WIPI1 puncta is cell context-dependent.

    “With regard to detection of the WIPI proteins, endogenous WIPI1 puncta cannot be detected in many cell types, 418 and the level of transiently expressed GFP-WIPI1 puncta is cell context-dependent; 418,419”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [418] source
    • cites [419] source

  • May combine two claims.

    Premisecitation

    Mutants such as atg1Δ accumulate Atg9 exclusively at the PAS, and this phenotype forms the basis of the TAKA assay.

    “Mutants such as atg1D accumulate Atg9 exclusively at the PAS, and this phenotype forms the basis of the transport of Atg9 after knocking out ATG1 (TAKA) assay.63”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] source

  • May combine two claims.

    Interpretationreasoning

    Monitoring changes in the activity of Atg1/ULK1 is not a direct assay for autophagy, although such changes may correlate with autophagy activity.

    “Nonetheless, it must be kept in mind that monitoring changes in the activity of Atg1/ ULK1 is not a direct assay for autophagy, although such changes may correlate with autophagy activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    GFP-WIPI1 and GFP-WIPI2 have been detected on the completed (mature) autophagosome by freeze-fracture analysis, but endogenous WIPI2 has not been detected on mRFP-LC3- or LAMP2-positive autophagosomes or autolysosomes using immunolabeling.

    “GFP-WIPI1 and GFP-WIPI2 have been detected on the completed (mature) autophagosome by freeze-fracture analysis, 60 but endogenous WIPI2 has not been detected on mRFP-LC3-or LAMP2-positive autophagosomes or autolysosomes using immunolabeling.418”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [60]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [418] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ATG9 displays partial colocalization with GFP-LC3.

    “ATG9 displays partial colocalization with GFP-LC3.396”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [396] source

  • May combine two claims.

    Interpretationnone given

    One disadvantage of stable transfectants is that the integration sites cannot always be predicted, and expression levels may not be optimal.

    “one disadvantage of stable transfectants is that the integration sites cannot always be predicted, and expression levels may not be optimal.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Stable transformants may have reduced background resulting from lower gene expression, and there is also the advantage of eliminating artifacts resulting from recent exposure to transfection reagents.

    “stable transformants may have reduced background resulting from the lower gene expression, and there is also the advantage of eliminating artifacts resulting from recent exposure to transfection reagents”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    These proteins bind phosphatidylinositol 3-phosphate (PtdIns3P) present at the phagophore and autophagosome and also PtdIns(3,5)P2.

    “These proteins bind phosphatidylinositol 3-phosphate (PtdIns3P) that is present at the phagophore and autophagosome 418,419 and also PtdIns(3,5)P 2 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [418] source
    • cites [419] source

  • May combine two claims.

    Premisecitation

    BECN1-GFP puncta detected by fluorescence microscopy or TEM may serve as an additional marker for autophagy induction; however, as with any GFP chimeras there is a concern that the GFP moiety interferes with correct localization of BECN1.

    “Thus, BECN1-GFP puncta detected by fluorescence microscopy or TEM may serve as an additional marker for autophagy induction; 436 however, as with any GFP chimeras there is a concern that the GFP moiety interferes with correct localization of BECN1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [436] source

  • May combine two claims.

    Interpretationreasoning

    Proteins other than Atg8/LC3 can be monitored to follow autophagy, and can be important tools to define specific steps of the process.

    “Conclusion: Proteins other than Atg8/LC3 can be monitored to follow autophagy, and can be important tools to define specific steps of the process.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the silkworm (B. mori) larval midgut, the occurrence of autophagy is accompanied by an upregulation of Atg5, Atg6 and Atg8 mRNA levels.

    “Similarly, in the silkworm (B.mori) larval midgut the occurrence of autophagy is accompanied by an upregulation of Atg5, Atg6 and Atg8 mRNA levels.452”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [452] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The gene that shows the greatest transcriptional regulation in the liver in response to starvation and circadian signals is Ulk1, but others also show more limited changes in mRNA levels including Gabarapl1, Bnip3 and, to a minor extent, Lc3B.

    “The gene that shows the greatest transcriptional regulation in the liver (in response to starvation and circadian signals) is Ulk1, but others also show more limited changes in mRNA levels including Gabarapl1, Bnip3 and, to a minor extent, Lc3B (Lin JD, personal communication).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lin JD, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    An upregulation of plant ATG8 may be needed during the adaptation to reproductive growth; a T-DNA inserted mutation of rice ATG8b blocked the change from vegetative growth to reproductive growth in both homozygous and heterozygous plant lines.

    “An upregulation of plant ATG8 may be needed during the adaptation to reproductive growth; a T-DNA inserted mutation of rice ATG8b blocked the change from vegetative growth to reproductive growth in both homozygous and heterozygous plant lines (Zhang M-Y, unpublished results).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Zhang M-Y, unpublished results

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Prolonged induction of the expression of ATG genes has been thought to allow the replenishment of critical proteins (e.g., LC3 and GABARAP) that are destroyed during autophagosome fusion with the lysosome.

    “Such prolonged induction of the expression of ATG genes has been thought to allow the replenishment of critical proteins (e.g., LC3 and GABARAP) that are destroyed during autophagosome fusion with the lysosome.459”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [459] source

  • May combine two claims.

    Interpretationreasoning

    It is not clear if these changes are sufficient to induce autophagy, and therefore these are not direct measurements.

    “It is not clear if these changes are sufficient to induce autophagy, however, and therefore these are not direct measurements.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Large changes in Atg gene transcription just prior to Drosophila salivary gland cell death, which is accompanied by an increase in autophagy, are detected in Atg2, Atg4, Atg5 and Atg7, whereas there is no significant change in Atg8a or Atg8b.

    “Of note, large changes in Atg gene transcription just prior to Drosophila salivary gland cell death (that is accompanied by an increase in autophagy) are detected in Atg2, Atg4, Atg5 and Atg7, whereas there is no significant change in Atg8a or Atg8b.449,450”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [449] source
    • cites [450] source

  • May combine two claims.

    Interpretationown data

    WIPI puncta formation can be used to monitor autophagy, but, similar to Atg8/LC3, should be examined in the presence and absence of inhibitors.

    “For example, WIPI puncta formation can be used to monitor autophagy, but, similar to Atg8/LC3, should be examined in the presence and absence of inhibitors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    The Pho8Δ60 assay requires standard positive and negative controls (such as an atg1Δ strain), and care must be taken to ensure the efficiency of cell lysis.

    “The Pho8D60 assay requires standard positive and negative controls (such as an atg1D strain), and care must be taken to ensure the efficiency of cell lysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Labeling for measuring degradation of long-lived proteins can be done with relative ease both in cultured cells and in live animals.

    “The labeling can be done with relative ease both in cultured cells, and in live animals.3”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] source

  • May combine two claims.

    Premisenone given

    In S. cerevisiae there are two genes encoding catalase activity, and only one of these gene products is localized in peroxisomes.

    “In S. cereviseae there are two genes encoding catalase activity, and only one of these gene products is localized in peroxisomes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Released amino acid may be further metabolized; branched chain amino acids are good indicators of proteolysis in hepatocytes, but not in muscle cells where they are further oxidized.

    “For example, branched chain amino acids are good indicators of proteolysis in hepatocytes, but not in muscle cells where they are further oxidized (Meijer AJ, personal communication).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Meijer AJ, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Study design and conductreasoning

    Because 3-MA could have some autophagy-independent effects in particular settings, it is advisable to verify that the 3-MA-sensitive degradation is also sensitive to general lysosomal inhibitors such as NH4Cl or leupeptin.

    “In addition, because 3-MA could have some autophagy-independent effects in particular settings it is advisable to verify that the 3-MA-sensitive degradation is also sensitive to general lysosomal inhibitors (such as NH 4 Cl or leupeptin).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    In a pulse-chase assay of specific protein turnover, a considerable fraction of the measured degradation will be non-autophagic, so it is important to also measure parallel cell samples treated with autophagy-suppressive concentrations of 3-MA or amino acids, or obtained from mutants missing central ATG components, and subtract these values from the total.

    “In this type of assay a considerable fraction of the measured degradation will be non-autophagic, and thus it is important to also measure, in parallel, cell samples treated with autophagysuppressive concentrations of 3-MA or amino acids, or obtained from mutants missing central ATG components (however, the latter assumes that non-autophagic proteolytic activity remains unchanged, which is unlike”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    Although there are changes in ATG gene expression that coincide with, and may be needed for, autophagy, this has not been carefully studied experimentally; therefore, at present the authors do not recommend monitoring ATG gene transcription as a general readout for autophagy unless there is clear documentation that the change(s) correlates with autophagy activity.

    “Conclusion: Although there are changes in ATG gene expression that coincide with, and may be needed for, autophagy, this has not been carefully studied experimentally.Therefore, at the present time we do not recommend the monitoring of ATG gene transcription as a general readout for autophagy unless there is clear documentation that the change(s) correlates with autophagy activity.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    It is generally better to follow protein levels because that is the ultimate readout that is significant with regard to the initiation and completion of autophagy, although ATG protein amounts do not always change significantly and the extent of increase is again cell type- and tissue-dependent.

    “In addition, it is generally better to follow protein levels because that is the ultimate readout that is significant with regard to the initiation and completion of autophagy, although ATG protein amounts do not always change significantly and the extent of increase is again cell type-and tissue-dependent.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    Under starvation conditions, prApe1 can enter the vacuole through nonspecific autophagy, and thus has been used as a marker for both the Cvt pathway and autophagy.

    “Under starvation conditions, prApe1 can enter the vacuole through nonspecific autophagy, and thus has been used as a marker for both the Cvt pathway and autophagy.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Changes in autophagy protein levels are not sufficient evidence of autophagy induction, and must be accompanied by additional assays as described herein.

    “Finally, changes in autophagy protein levels are not sufficient evidence of autophagy induction, and must be accompanied by additional assays as described herein.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    The yeast Cvt pathway is unique in that it is a biosynthetic route that utilizes the autophagy-related protein machinery, whereas other types of selective autophagy are degradative.

    “The yeast Cvt pathway is unique in that it is a biosynthetic route that utilizes the autophagy-related protein machinery, whereas other types of selective autophagy are degradative.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    Inclusion of the appropriate unlabeled amino acid in the starvation medium at a concentration equivalent to that of other amino acids in the chase medium is necessary; otherwise the released [14C]-amino acid is effectively re-incorporated into cellular proteins, resulting in a significant underestimation of protein degradation.

    “Note that the inclusion of the appropriate unlabeled amino acid (i.e., valine, leucine or methionine) in the starvation medium at a concentration equivalent to that of other amino acids in the chase medium is necessary; otherwise, the released [ 14 C]-amino acid is effectively re-incorporated into cellular proteins, which results in a significant underestimation of protein degradation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Study design and conductreasoning

    It is important to determine that the marker is distributed homogeneously in the cytoplasm.

    “Consequently, it is important to determine that the marker is distributed homogeneously in the cytoplasm.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Selective autophagy types include pexophagy, mitophagy, reticulophagy, ribophagy and xenophagy, and each process has its own marker proteins, although these are typically variations of other assays used to monitor the Cvt pathway or autophagy.

    “The latter include pexophagy, mitophagy, reticulophagy, ribophagy and xenophagy, and each process has its own marker proteins, although these are typically variations of other assays used to monitor the Cvt pathway or autophagy.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    To minimize background activity in the Pho8Δ60 assay, it is preferable to have the gene encoding cytosolic alkaline phosphatase (PHO13) additionally deleted.

    “To minimize background activity, it is preferable to have the gene encoding cytosolic alkaline phosphatase (PHO13) additionally deleted (although this is not necessary when assaying certain substrates).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Study design and conductnone given

    When using inhibitors it is critical to know whether the inhibitors being used alter autophagy in the particular cell type and context being examined.

    “Thus, when using inhibitors it is critical to know whether the inhibitors being used alter autophagy in the particular cell type and context being examined.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Compensation between degradative pathways might interfere with control measurements under conditions that attempt to inhibit macroautophagy; however, as macroautophagy is the major degradative pathway, the contributions of other types of degradation over the course of this type of experiment are most often negligible.

    “This compensation might interfere with control measurements under conditions that attempt to inhibit macroautophagy; however, as the latter is the major degradative pathway, the contributions of other types of degradation over the course of this type of experiment are most often negligible.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    Protein degradation assays represent a well-established methodology for measuring autophagic flux, and they allow good quantification.

    “Protein degradation assays represent a well-established methodology for measuring autophagic flux, and they allow good quantification.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    Monitoring changes in mRNA levels for either ATG genes or autophagy regulators may provide some evidence for autophagy induction, but should be used along with other methods.

    “Thus, monitoring changes in mRNA levels for either ATG genes or autophagy regulators may provide some evidence for autophagy induction, but should be used along with other methods.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    PARK2 translocates to damaged mitochondria and ubiquitinates a wide range of outer membrane proteins including VDAC1, MFN1/2 and TOMM20.

    “PARK2 translocates to damaged mitochondria and ubiquitinates a wide range of outer membrane proteins including VDAC1, MFN1/2 and TOMM20.547,[555][556][557]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 547,[555][556][557]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    There is no consensus at the present time with regard to the best method for monitoring mammalian mitophagy.

    “There is no consensus at the present time with regard to the best method for monitoring mammalian mitophagy.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Some of the mutated dysferlin protein, LGMB2B/Miyoshi type muscle dystrophy, which accumulates in the ER, is degraded by ER-stress mediated reticulophagy.

    “Some of the mutated dysferlin protein, LGMB2B/Miyoshi type muscle dystrophy, which accumulates in the ER, is degraded by ER-stress mediated reticulophagy.528”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [528] source

  • May combine two claims.

    Interpretationnone given

    The GFP-Atg8 protease protection assay avoids the problems of prApe1, but its signal-to-noise ratio is typically substantially higher.

    “The GFP-Atg8 protease protection assay avoids these problems, but the signal-to-noise ratio is typically substantially higher.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The GFP-Atg8, or similar, processing assay is easy to perform and is suitable for analysis by microscopy as well as western blotting; however, particular care is needed to obtain quantitative data for GFP-Atg8, Pex14-GFP or Om45-GFP, etc. processing assays.

    “The GFP-Atg8, or similar, processing assay is easy to perform and is suitable for analysis by microscopy as well as western blotting; however, particular care is needed to obtain quantitative data for GFP-Atg8, Pex14-GFP or Om45-GFP, etc. processing assays”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisereasoning

    Usually 6 h of mitophagy induction is needed to be able to detect free GFP (e.g., from Om45-GFP) by western blot under starvation conditions, whereas stationary phase mitophagy typically requires 3 d before a free GFP band is observed.

    “Usually 6 h of mitophagy induction is needed to be able to detect free GFP (e.g., from Om45-GFP) by western blot under starvation conditions, whereas stationary phase mitophagy typically requires 3 d before a free GFP band is observed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Particular mutants may display complete maturation of prApe1 under autophagy-inducing conditions, but may still have a defect in other types of selective autophagy, as well as being unable to induce a normal level of nonspecific autophagy.

    “Accordingly, particular mutants may display complete maturation of prApe1 under autophagy-inducing conditions, but may still have a defect in other types of selective autophagy, as well as being unable to induce a normal level of nonspecific autophagy.63”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [63] source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    The Cvt pathway has been demonstrated to occur only in yeast.

    “The Cvt pathway has been demonstrated to occur only in yeast.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Overexpression of prApe1 saturates import by the Cvt pathway, and the precursor form accumulates, but is rapidly matured upon autophagy induction.

    “Overexpression of prApe1 saturates import by the Cvt pathway, and the precursor form accumulates, but is rapidly matured upon autophagy induction.218”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [218] source

  • May combine two claims.

    Premisenone given

    In yeast, nonspecific autophagy can be induced by nitrogen starvation conditions, whereas degradative types of selective autophagy generally require a carbon source change or ER stress for efficient induction.

    “In yeast, nonspecific autophagy can be induced by nitrogen starvation conditions, whereas degradative types of selective autophagy generally require a carbon source change or ER stress for efficient induction.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Reticulophagy-associated autophagosomes are lamellar-membraned structures that contain ER proteins.

    “Reticulophagyassociated autophagosomes are lamellar-membraned structures that contain ER proteins.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In yeast, gluconeogenic enzymes such as fructose-1,6-bisphosphatase (Fbp1, also called FBPase), malate dehydrogenase (Mdh2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) constitute the cargo of the vacuole import and degradation (Vid) pathway.

    “In yeast, gluconeogenic enzymes such as fructose-1,6-bisphosphatase (Fbp1, also called FBPase), malate dehydrogenase (Mdh2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) constitute the cargo of the vacuole import and degradation (Vid) pathway.533”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [533] source

  • May combine two claims.

    Premisecitation

    prApe1 cannot be used as an autophagy reporter for mutants that are not defective in the Cvt pathway, although this can be bypassed by using a vac8D background.

    “Another problem is that prApe1 cannot be used as an autophagy reporter for mutants that are not defective in the Cvt pathway, although this can be bypassed by using a vac8D background.603”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [603] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nucleus-vacuole junctions (NVJs) are generated by interaction of the outer nuclear membrane protein Nvj1 with the vacuolar protein Vac8.

    “These nucleus-vacuole junctions (NVJs) are generated by interaction of the outer nuclear membrane protein Nvj1 with the vacuolar protein Vac8.523”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [523] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    To assure that the measured PMN rate is indeed due to selective micronucleophagy, appropriate controls such as cells lacking Nvj1 or Vac8 should be included.

    “To assure that the measured PMN rate is indeed due to selective micronucleophagy, appropriate controls such as cells lacking Nvj1 or Vac8 should be included.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    When working with yeast it is preferable to use strains that have the marker proteins integrated into the chromosome rather than relying on plasmid-based expression, because plasmid numbers can vary from cell to cell.

    “In general, when working with yeast it is preferable to use strains that have the marker proteins integrated into the chromosome rather than relying on plasmid-based expression, because plasmid numbers can vary from cell to cell.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Autophagy is also used for the selective removal of ribosomes.

    “Autophagy is also used for the selective removal of ribosomes.529”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [529] source

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Study design and conductreasoning

    It is best to monitor more than one protein, and to include an inner membrane or matrix component in the analysis.

    “It is best to monitor more than one protein, and to include an inner membrane or matrix component in the analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Study design and conductreasoning

    It is good practice to evaluate autophagosome size and number by TEM.

    “For this reason, it is good practice to evaluate autophagosome size and number by TEM.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    It is best to use a strain background that is pep4D vps4D to prevent the breakdown of the autophagic bodies, and to eliminate confounding vesicles from the multivesicular body pathway.

    “It is best to use a strain background that is pep4D vps4D to prevent the breakdown of the autophagic bodies, and to eliminate confounding vesicles from the multivesicular body pathway.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    It is much simpler to monitor autophagic bodies rather than autophagosomes in yeast.

    “Actually, it is much simpler to monitor autophagic bodies (rather than autophagosomes) in yeast.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisereasoning

    PMN vesicles contain nuclear material and are limited by three membranes with the outermost derived from the vacuole, and the two inner ones from the nuclear ER.

    “PMN vesicles thus contain nuclear material and are limited by three membranes with the outermost derived from the vacuole, and the two inner ones from the nuclear ER.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    At present no publications report use of the GFP-Atg8 protease protection assay other than in yeast.

    “In theory, it should be possible to use this assay in other cell types, but at present no publications report its use other than in yeast.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Study design and conductnone given

    As with any organelle-specific form of autophagy, it is necessary to demonstrate increased levels of autophagosomes containing mitochondria, maturation of these autophagosomes to mitochondrial degradation that can be blocked by specific inhibitors of autophagy or lysosomal degradation, and whether the changes are due to selective mitophagy or increased mitochondrial degradation during generalized.

    “As with any organelle-specific form of autophagy, it is necessary to demonstrate: (1) increased levels of autophagosomes containing mitochondria, (2) maturation of these autophagosomes to culminate with mitochondrial degradation, which can be blocked by specific inhibitors of autophagy or of lysosomal degradation, and (3) whether the changes are due to selective”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Piecemeal microautophagy of the nucleus (PMN) is another selective autophagic subtype, which targets portions of the nucleus for degradation.

    “Piecemeal microautophagy of the nucleus (PMN, also micronucleophagy) is another selective autophagic subtype, which targets portions of the nucleus for degradation.[520][521][522]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [520][521][522]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    The peak of the excitation spectrum of the Keima protein shifts from 440 nm to 586 nm when mitochondria are delivered to acidic lysosomes, which allows easy quantification of mitophagy.

    “The peak of the excitation spectrum of the protein shifts from 440 nm to 586 nm when mitochondria are delivered to acidic lysosomes, which allows easy quantification of mitophagy (Fig. 22).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Autophagic bodies may coalesce in the vacuole lumen, making it difficult to obtain an accurate quantification.

    “One caveat to the detection of autophagic bodies, however, is that they may coalesce in the vacuole lumen, making it difficult to obtain an accurate quantification.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    For the GFP-Atg8 processing assay, 2 h of starvation is generally sufficient to detect a significant level of free (i.e., vacuolar) GFP by western blotting as a measure of nonselective autophagy.

    “In addition, for the GFP-Atg8 processing assay, 2 h of starvation is generally sufficient to detect a significant level of free (i.e., vacuolar) GFP by western blotting as a measure of nonselective autophagy.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Study design and conductreasoning

    It is advisable to use a variety of complementary methods to monitor mitochondria loss including TEM, single cell analysis of LC3 fluorescent puncta, and western blot, in conjunction with flux inhibitors and specific inhibitors of autophagy induction compared with inhibitors of the other major degradation systems.

    “Thus, it is advisable to use a variety of complementary methods to monitor mitochondria loss including TEM, single cell analysis of LC3 fluorescent puncta, and western blot, in conjunction with flux inhibitors and specific inhibitors of autophagy induction compared with inhibitors of the other major degradation systems”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The turnover of LC3-II or rate of basal autophagic flux is not known for tissues in vivo, and therefore short treatments (e.g., 4 h) may not be as effective as blocking for longer times (e.g., 12 to 24 h).

    “The turnover of LC3-II or rate of basal autophagic flux is not known for tissues in vivo, and therefore short treatments (e.g., 4 h) may not be as effective as blocking for longer times (e.g., 12 to 24 h).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Not all lysosomes are alike; there are differences among primary lysosomes, autolysosomes and telolysosomes.

    “Finally, it is worthwhile noting that not all lysosomes are alike.For example, there are differences among primary lysosomes, autolysosomes and telolysosomes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Monitoring autophagic flux in vivo or in organs is one of the least developed areas at present, and ideal methods relative to cell culture may not exist.

    “Monitoring autophagic flux in vivo or in organs is one of the least developed areas at present, and ideal methods relative to the techniques possible with cell culture may not exist.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Basal autophagy or sensitivity to autophagic induction may vary with animal age, sex or strain background.

    “Moreover, basal autophagy or sensitivity to autophagic induction may vary with animal age, sex or strain background.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Because lysosomes are able to take up sucrose if present at high concentrations, the use of sucrose gradients for isolation of intact lysosome-related organelles is strongly discouraged.

    “In that respect, because lysosomes are able to take up sucrose if it is present at high concentrations, the use of sucrose gradients for the isolation of intact lysosome-related organelles is strongly discouraged.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    GFP-LC3 transgenic mice exhibit prominent fluorescence in podocytes but rarely in tubular epithelial cells in the kidney, and a similar GFP pattern is observed in transgenic mice carrying CAG promoter-driven non-tagged GFP.

    “For example, GFP-LC3 transgenic mice exhibit prominent fluorescence in podocytes, but rarely in tubular epithelial cells in the kidney, 126 but a similar GFP pattern is observed in transgenic mice carrying CAG promoter-driven non-tagged GFP.655”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [126] source
    • cites [655] source

  • May combine two claims.

    Interpretationreasoning

    The duration of turnover experiments is usually only a few hours, so long-term side effects or declining effectiveness of inhibitors can be avoided.

    “The duration of such experiments is usually only a few hours; therefore, long-term side effects or declining effectiveness of the inhibitors can be avoided.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Any autophagic blocker (leupeptin, bafilomycin A1, chloroquine, or microtubule depolarizing agents such as colchicine or vinblastine) must significantly increase basal LC3-II levels.

    “Any autophagic blocker, whether leupeptin, bafilomycin A 1 , chloroquine or microtubule depolarizing agents such as colchicine or vinblastine must significantly increase basal LC3-II levels.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Any part of such a fraction can be considered a representative sample of tissue constituents and used in quantitative biochemical, centrifugational and morphological studies of autophagic particle populations.

    “Any part of such a fraction can be considered to be a representative sample of tissue constituents and used in quantitative biochemical, centrifugational and morphological studies of autophagic particle populations.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Cycloheximide is sometimes used as an autophagy inhibitor, but this is problematic because of many potential indirect effects.

    “Cycloheximide is sometimes used as an autophagy inhibitor, but this is problematic because of the many potential indirect effects.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Through homogenization and resuspension, complex and heterogeneous components of subcellular fractions become randomly distributed throughout the fraction volume; therefore, any aliquot of that volume can be considered a random sample of the whole volume.

    “However, through homogenization and resuspension, complex and heterogeneous components of subcellular fractions become randomly distributed throughout the fraction volume.Therefore, any aliquot of that volume can be considered a random sample of the whole volume.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The relative contribution of autophagy to post-absorptive release of amino acids may be changed in disease states.

    “However, the relative contribution of autophagy to the post-absorptive release of amino acids may be changed in disease states (see cautionary notes).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Acid hydrolases are found primarily in membrane-bound compartments and their latent activities cannot be measured unless these membranes are lysed.

    “for example, acid hydrolases are found primarily in membrane-bound compartments and their latent activities cannot be measured unless these membranes are lysed”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Treatment with cycloheximide causes a potent increase in TORC1 activity, which can decrease autophagy, in part as a result of the increase in the amino acid pool resulting from suppressed protein synthesis.

    “Treatment with cycloheximide causes a potent increase in TORC1 activity, which can decrease autophagy, in part as a result of the increase in the amino acid pool resulting from suppressed protein synthesis”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [606] source
    • cites [607] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Transglutaminase type 2 protein levels decrease in mouse liver in vivo upon starvation in an autophagy-dependent manner.

    “and a study shows that transglutaminase type 2 protein levels decrease in mouse liver in vivo upon starvation in an autophagy-dependent manner (and in human cell lines in vitro in response to various stimuli; Piacentini M, personal communication)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Many inhibitors such as bafilomycin A1 or chloroquine have relatively poor bioavailability to the central nervous system.

    “For example, many inhibitors such as bafilomycin A 1 or chloroquine have relatively poor bioavailability to the central nervous system.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    Teflon/glass motor homogenization is suitable for tissues with abundant connective tissue such as liver, but for circulating cells or cells in culture, disruption by nitrogen cavitation is a good method to preserve lysosomal membrane stability; however, this method is not suitable for small samples and may not be readily available.

    “For example teflon/glass motor homogenization is suitable for tissues with abundant connective tissue, such as liver, but for circulating cells or cells in culture, disruption by nitrogen cavitation is a good method to preserve lysosomal membrane stability; 625 however, this method is not suitable for small samples and may not be readily available.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [625] source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premisenone given

    What we refer to as lysosomes are actually a very heterogeneous pool of organelles that fulfill five classical criteria: pH < 5.6, mature cathepsins, presence of LAMP proteins, a single membrane, and absence of endosomal and recycling compartment markers.

    “Furthermore, what we refer to as “lysosomes” are actually a very heterogeneous pool of organelles that simply fulfill five classical criteria, having a pH , 5.6, mature cathepsins, the presence of LAMP proteins, a single membrane, and the absence of endosomal and recycling compartment markers”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    Some autophagic substrates can accumulate with either an increase or a decrease in autophagic flux, so their presence or absence needs cautious interpretation.

    “however, their presence or absence needs to be cautiously interpreted as some of these substrates can accumulate with either an increase or a decrease in autophagic flux”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    β-hexosaminidase is routinely used to assess lysosomal purity, but enzymes such as CTSB/cathepsin B may also be used and may provide more accurate readouts.

    “of enzymes or protein markers for those compartments (e.g., β-hexosaminidase is routinely used to assess lysosomal purity, but enzymes such as CTSB/cathepsin B may also be used and may provide more accurate readouts).625”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [625] source

  • May combine two claims.

    Premisecitation

    At high concentrations (millimolar range) cycloheximide inhibits complex I of the mitochondrial respiratory chain, but this is not a problem, at least in hepatocytes, at low concentrations (10-20 mM) sufficient to prevent protein synthesis.

    “In addition, at high concentrations (in the millimolar range) cycloheximide inhibits complex I of the mitochondrial respiratory chain, 608,609 but this is not a problem, at least in hepatocytes, at low concentrations (10 -20 mM) that are sufficient to prevent protein synthesis”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [608] source
    • cites [609] source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Treating animals with bafilomycin A1 for 2 h can be quite toxic.

    “The toxicity of the blocking agent needs to be considered (e.g., treating animals with bafilomycin A 1 for 2 h can be quite toxic)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Amino acid exchange studies show that peripheral tissues take up amino acids during the post-prandial (fed) state and release amino acids in the postabsorptive (fasted) state.

    “Amino acid exchange studies show that the peripheral tissues take up amino acids during the post-prandial (fed) state and release amino acids in the postabsorptive (fasted) state, i.e., in a state with relatively low plasma insulin and amino acid levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    GFP-LC3 expression is driven by the CAG promoter, so intensity of GFP signal may not always represent actual autophagic activity but rather CAG promoter activity in individual cells.

    “When analyzing basal autophagic level in vivo using GFP-LC3 transgenic mice, 126 one pitfall is that GFP-LC3 expression is driven by the cytomegalovirus enhancer and β-actin (CAG) promoter, so that the intensity of the GFP signal may not always represent the actual autophagic activity, but rather the CAG promoter activity in individual cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [126] source

  • May combine two claims.

    Premisecitation

    From each 0.1 ml of pellet volume, 2,000,000 sections can be obtained, so any practical sample size is an infinitesimally small subsample of the total sample.

    “With the latter, 2,000,000 sections can be obtained from each 0.1 ml of pellet volume, so any practical sample size is an infinitesimally small subsample of the total sample.618”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [618]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    Differential centrifugation can be used to obtain a subcellular fraction enriched in mitochondria and organelles of the autophagic-lysosomal system, which can then be subjected to density gradient centrifugation to enrich autophagosomes, amphisomes, autolysosomes and lysosomes.

    “Because of their sizes [smaller than nuclei but larger than membrane fragments (microsomes)], differential centrifugation can be used to obtain a subcellular fraction enriched in mitochondria and organelles of the autophagic-lysosomal system, which can then be subjected to density gradient centrifugation to enrich autophagosomes, amphisomes, autolysosomes and lysosomes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In U87MG cells treated with tamoxifen, BECN1 does not appear to be upregulated, whereas the levels of LC3-II and SQSTM1 are increased, while LAMP2B is downregulated and CTSD and CTSL1 activities are almost completely blocked.

    “However, in U87MG cells treated with tamoxifen BECN1 does not appear to be upregulated, whereas the levels of LC3-II and SQSTM1 are increased, while LAMP2B is downregulated and CTSD and CTSL1 activities are almost completely blocked (Choi KS, personal communication).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Choi KS, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The term autophagic cell death was coined in the 1970s in a purely morphological context to refer to cell death with autophagic features, and death-mediation was not part of the definition.

    “Other papers suffer from ambiguous use of the term “autophagic cell death,” which was coined in the 1970s 667 in a purely morphological context to refer to cell death with autophagic features (especially the presence of numerous secondary lysosomes); this was sometimes taken to suggest a role of autophagy in the cell death mechanism, but death-mediation was not part of the definition.668”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [667] source
    • cites [668] source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    Autophagy may be involved not only in allowing this death to occur but also in the vacuolization process itself.

    “Autophagy may be involved not only in allowing this death to occur, but also in the vacuolization process itself.674”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [674] source

  • May combine two claims.

    Interpretationcitation

    Claim (c) is stronger than claim (b) because proof is needed that the cell death mechanism is not apoptosis or necrosis.

    “Claim (c) is still stronger, because, even if the cell death is blocked by autophagy inhibition, proof needs to be provided that the cell death mechanism is not apoptosis or necrosis.670”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [670] source

  • May combine two claims.

    Interpretationnone given

    Accumulation of ubiquitin may indicate a block in autophagy, inhibition of proteasomal degradation, or structural changes in substrate proteins that hinder degradation.

    “One caution in using approaches that monitor ubiquitinated aggregates is that the accumulation of ubiquitin may indicate a block in autophagy, inhibition of proteasomal degradation, or may correspond to structural changes in the substrate proteins that hinder their degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    Although morphology of vacuolar cell death resembles macro- and microautophagy, there is no genetic evidence yet that this death requires the core autophagic machinery.

    “Although the morphology of vacuolar cell death resembles a combination of macro-and microautophagy, there is no genetic evidence yet that this death requires the core autophagic machinery.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    In the central nervous system, absence of autophagy leads to rapid degeneration.

    “This type of control will be impractical for some tissues such as those of the central nervous system because the absence of autophagy leads to rapid degeneration.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The absence of autophagy leads to rapid degeneration in the central nervous system.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisereasoning

    The term autophagic cell death is now used in at least three different ways: autophagy-associated cell death, autophagy-mediated cell death, and a distinct mechanism of cell death independent of apoptosis or necrosis.

    “Unfortunately, the term “autophagic cell death” is now used in at least three different ways: (a) autophagyassociated cell death (the original meaning); (b) autophagymediated cell death (which could involve a standard mechanism of cell death such as apoptosis, but triggered by autophagy); (c) a distinct mechanism of cell death, independent of apoptosis or necrosis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Only cytosolic and not nuclear ubiquitin is subject to autophagic degeneration.

    “In addition, only cytosolic and not nuclear ubiquitin is subject to autophagic degeneration.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tamoxifen acts to induce autophagy by increasing the expression of BECN1 in MCF7 cells.

    “Relatively little is known about direct regulation via the ATG proteins, but there is some indication that tamoxifen acts to induce autophagy by increasing the expression of BECN1 in MCF7 cells.767”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [767]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Insulin has no effect on proteolysis in suspended rat hepatocytes, in contrast to the result with perfused rat liver.

    “For example, insulin has no effect on proteolysis in suspended rat hepatocytes, in contrast to the result with perfused rat liver.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [780]
    • cites [781] source

  • May combine two claims.

    Interpretationreasoning

    The levels of basal autophagy can vary substantially and can mask the effects of the experimental parameters being tested.

    “Conclusion: The levels of basal autophagy can vary substantially and can mask the effects of the experimental parameters being tested.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Basal levels of LC3-II or GFP-LC3 puncta may change according to the time after addition of fresh medium to cells.

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    Although autophagy is conserved from yeast to human, there may be tremendous differences in the specific details among systems.

    “Conclusion: Although autophagy is conserved from yeast to human, there may be tremendous differences in the specific details among systems.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Autophagy is conserved from yeast to human.

  • May combine two claims.

    Interpretationnone given

    Rapamycin is much less effective at inhibiting MTOR and inducing autophagy than catalytic inhibitors.

    “Conclusion: Rapamycin is much less effective at inhibiting MTOR and inducing autophagy than catalytic inhibitors, and the latter should therefore be considered for use instead of rapamycin; however, it must be kept in mind that catalytic inhibitors also affect MTORC2.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The insulin effect reappears when isolated hepatocytes are incubated in stationary dishes or are allowed to settle down on the matrix.

    “The insulin effect reappears, however, when isolated hepatocytes are incubated in stationary dishes 780,781 or are allowed to settle down on the matrix (Häussinger D, personal communication).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [780]
    • cites [781] source
    • cites Häussinger D, personal communication

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Autophagy regulation by insulin and some amino acids might require volume sensing via integrin-matrix interactions and also intact microtubules.

    “The reason for this might be that autophagy regulation by insulin and some amino acids requires volume sensing via integrin-matrix interactions and also intact microtubules.[782][783][784]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [782][783][784]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    If drugs are added after a time in which basal autophagy is already high, then the effects of the drug can be masked by the cell's basal autophagy, and wrong conclusions may be drawn.

    “If the drugs are added after a time in which basal autophagy is already high, then the effects of the drug can be masked by the cell's basal autophagy, and wrong conclusions may be drawn.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Basal levels of LC3-II or GFP-LC3 puncta may change according to the time after addition of fresh medium to cells.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Results based on one system should not be assumed to be applicable to another.

    “Thus, results based on one system should not be assumed to be applicable to another.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Autophagy is conserved from yeast to human.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    C. elegans has a single ortholog of most yeast Atg proteins; however, two nematode homologs exist for Atg4 and Atg8.

    “C. elegans has a single ortholog of most yeast Atg proteins; however, two nematode homologs exist for Atg4 and Atg8.785”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [785] source
225 of 225

  • The authors state this as an assumption.

    In methods that arrest autophagic flux at a given point and record time-dependent accumulation of a marker, there is no feedback of the accumulating structure on its own rate of formation.

    “however, the latter assumes there is no feedback of the accumulating structure on its own rate of formation.23”

  • The paper relies on this without support.

    Prolonged inhibition of lysosomal hydrolases (>6 h) is likely to induce a secondary autophagic response triggered by the accumulated undigested autophagy cargo.

    “Prolonged inhibition of lysosomal hydrolases (. 6 h) is likely to induce a secondary autophagic response triggered by the accumulated undigested autophagy cargo.”

  • The paper relies on this without support.

    Endogenous ULK1 is of low abundance in many systems, making it difficult to detect phospho-ULK1 by western blot analysis.

    “One problem in monitoring assembly of the ULK1 complex is the low abundance of endogenous ULK1 in many systems, which makes it difficult to detect phospho-ULK1 by western blot analysis.”

  • The paper relies on this without support.

    There is not a complete consensus on the specific residues of ULK1 that are targeted by AMPK or MTOR.

    “Finally, there is not a complete consensus on the specific residues of ULK1 that are targeted by AMPK or MTOR.”

  • The paper relies on this without support.

    Apparently contradictory data have been published regarding the association of AMPK and MTOR with the ULK1 kinase complex under different conditions.

    “Similarly, apparently contradictory data have been published regarding the association of AMPK and MTOR with the ULK1 kinase complex under different conditions.”

  • The paper relies on this without support.

    A good antibody that can be used to detect endogenous ATG14 does not exist.

    “Currently, a good antibody that can be used to detect the endogenous ATG14 does not exist.”

  • The paper relies on this without support.

    Most of the ATG genes do not show significant changes in mRNA levels when autophagy is induced.

    “Cautionary notes: Most of the ATG genes do not show significant changes in mRNA levels when autophagy is induced.”

  • The paper relies on this without support.

    In any cell culture system different types of cells (for example, those undergoing autophagy or not) exist simultaneously.

    “Another general caution pertains to the fact that in any cell culture system different types of cells (for example, those undergoing autophagy or not) exist simultaneously.”

  • The paper relies on this without support.

    Only an average level of protein or mRNA expression can be evaluated with most methods.

    “Therefore, only an average level of protein or mRNA expression can be evaluated with most methods.”

  • The paper relies on this without support.

    Mammalian cell lines may have mutations that alter autophagy signaling or execution.

    “Finally, researchers need to realize that mammalian cell lines may have mutations that alter autophagy signaling or execution; this problem can be avoided by using primary cells.”

  • The paper relies on this without support.

    If autophagic protein degradation is low, it may be difficult to measure it reliably above the relatively high background of non-autophagic degradation.

    “If the autophagic protein degradation is low (as it will be in cells in replete medium), it may be difficult to measure it reliably above the relatively high background of non-autophagic degradation.”

  • The paper relies on this without support.

    Mice are nocturnal animals, so they preferentially move and eat during the night, while they mostly rest during daylight.

    “Mice are nocturnal animals, so they preferentially move and eat during the night, while they mostly rest during daylight.”

  • The paper relies on this without support.

    The absence of autophagy leads to rapid degeneration in the central nervous system.

    “because the absence of autophagy leads to rapid degeneration.”

  • The paper relies on this without support.

    Autophagy is an essential metabolic process for many cell types and tissues.

    “since autophagy is an essential metabolic process for many cell types and tissues, loss of viability due to autophagy inhibition always has to be a concern when analyzing cell death-unrelated questions.”

  • The paper relies on this without support.

    The long-term secondary consequences of gene knockouts or knockdowns are likely much more complex than the immediate effects of the actual autophagy inhibition.

    “Finally, we note that the long-term secondary consequences of gene knockouts or knockdowns are likely much more complex than the immediate effects of the actual autophagy inhibition.”

  • The paper relies on this without support.

    Viral autophagy inhibitor proteins are used for the regulation of usually more than one cellular pathway by the respective pathogens.

    “because these proteins are used for the regulation of usually more than one cellular pathway by the respective pathogens.”

  • The paper relies on this without support.

    A shift to a new medium can be considered a stress situation.

    “A shift to a new medium can be considered a stress situation.”

  • The paper relies on this without support.

    It is not possible to state explicit rules that can be applied to all experimental systems.

    “Throughout these guidelines we have noted that it is not possible to state explicit rules that can be applied to all experimental systems.”

  • The paper relies on this without support.

    Some techniques may not work in particular cell types or organisms.

    “For example, some techniques may not work in particular cell types or organisms.”

  • The paper relies on this without support.

    Differences may be seen between in vivo or perfused organ studies and cell culture analyses.

    “Differences may also be seen between in vivo or perfused organ studies and cell culture analyses.”

  • The paper relies on this without support.

    Autophagy is conserved from yeast to human.

    “Although autophagy is conserved from yeast to human, there may be tremendous differences in the specific details among systems.”

  • The paper relies on this without support.

    The main concern with pharmacological manipulations is pleiotropic effects of the compound being used.

    “The main concern with pharmacological manipulations is pleiotropic effects of the compound being used.”

  • The paper relies on this without support.

    Catalytic inhibitors also affect MTORC2.

    “however, it must be kept in mind that catalytic inhibitors also affect MTORC2.”

  • The paper relies on this without support.

    Basal levels of LC3-II or GFP-LC3 puncta may change according to the time after addition of fresh medium to cells.

    “Basal levels of LC3-II or GFP-LC3 puncta may change according to the time after addition of fresh medium to cells, and this can lead to misinterpretations of what basal autophagy means.”
24 of 24

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Most studies of autophagy in the nervous system are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations.

    “most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations”

    From Measurement of autophagy flux in the nervous system in vivo (2013) · cites this paper as [4]

    The Introduction and the passage headed “Do not distribute” (page unavailable) explain that static autophagosome measurements can be difficult to interpret without measuring autophagic flux. The in-vivo analyses section (page unavailable) notes limitations of marker staining relevant to neurodegeneration, but the cited passages do not establish that most nervous-system studies use such measurements or that this has led to conflicting interpretations. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

  • Accurate methods to measure autophagic activity in vivo in neurons are not available.

    “Accurate methods to measure autophagic activity in vivo in neurons are not available”

    From Measurement of autophagy flux in the nervous system in vivo (2013) · cites this paper as [4]

    The “Analyses in vivo” section says that monitoring autophagic flux in vivo is underdeveloped and that ideal methods may not exist. It does not establish that accurate methods are unavailable specifically for neurons; its examples of in vivo monitoring concern other tissues, while its brain-slice example is ex vivo. No page number is available in our copy. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • The mCherry-GFP-LC3 reporter is designed to monitor LC3 flux because GFP signal is sensitive to acidic and/or proteolytic conditions of the lysosome lumen, whereas mCherry fluorescence persists under these environments.

    “This fluorescent reporter is designed to monitor LC3 flux through the autophagy pathway because the GFP signal is sensitive to the acidic and/or proteolytic conditions of the lysosome lumen, whereas mCherry fluorescence persists under these environments.4, 17”

    From Measurement of autophagy flux in the nervous system in vivo (2013) · cites this paper as [4]

  • Monitoring static autophagy markers can lead to misleading interpretations.

    “Because monitoring these markers can lead to misleading interpretations,4 there is a growing need for simple methods to measure autophagy flux in neurons in vivo.”

    From Measurement of autophagy flux in the nervous system in vivo (2013) · cites this paper as [4]

  • Chloroquine treatment does not cause accumulation of LC3-II in Atg5ΔRPE RPE.

    “Upon treatment with chloroquine to block autophagic flux (Klionsky et al., 2012), RPE still failed to accumulate LC3-II in Atg5ΔRPE mice (Figure 1C) verifying the loss of Atg5.”

    From Noncanonical autophagy promotes the visual cycle (2013) · cites this paper as Klionsky et al., 2012

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • GABARAPL3 is both included in and excluded from the GABARAP family

    Minor

    The initial description lists GABARAPL3 as a member of the four-protein GABARAP subfamily, but a later passage says it should not be considered the family's fourth member.

1 of 1

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1073/pnas.1113421108

    Info

    The cited work was retracted on 2025-08-04 (notice 10.1073/pnas.2518460122), not before this paper was published (2012-04).

    Cited work 10.1073/pnas.1113421108 · retracted 2025-08-04 · notice 10.1073/pnas.2518460122 · this paper published 2012-04

1 of 1

Cites

1 retracted

Flagged first, then by citations

Research works printed but not linked: 23

  1. Seglen PO. Regulation of autophagic protein degradation in isolated liver cells. In: Glaumann H and Ballard FJ, eds. Lysosomes: Their Role in Protein Breakdown. London: Academic Press, 1987:369-414.
  2. Kovács J, 1986, Biomed Biochim Acta, 45, 1543
  3. Seglen PO, 1981, Acta Biol Med Ger, 40, 1587
  4. Klionsky DJ. Autophagy: Lower Eukaryotes and Non-Mammalian Systems, Part A. Amsterdam: Academic Press/Elsevier, 2008.
  5. Klionsky DJ. Autophagy in Mammalian Systems, Part B. Amsterdam: Academic Press/Elsevier, 2008.
  6. Réz G, 1980, Lab Invest, 43, 269
  7. Kovács J, 1982, Acta Biol Med Ger, 41, 131
  8. Hirsimäki Y, 1982, Eur J Cell Biol, 27, 298
  9. Williams MA. Quantitative methods in biology: Practical methods in electron microscopy. Amsterdam, New York, Oxford: North-Holland Publishing Company, 1977.
  10. Shelburne JD, 1973, Am J Pathol, 73, 641
  11. Réz G, 1991, Acta Biol Hung, 42, 133
  12. Réz G, 1996, Eur J Cell Biol, 71, 341
  13. 10.1126/science.1101738 · DOI · Paper page
  14. Yokota S, 1993, Eur J Cell Biol, 61, 67
  15. Biolo G, 1995, Am J Physiol, 268, E75
  16. Denne SC, 1991, Am J Physiol, 261, E809
  17. Paglin S, 2001, Cancer Res, 61, 439
  18. Biederbick A, 1995, Eur J Cell Biol, 66, 3
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Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J. A., Ahn, H. J., Ait-Mohamed, O., Ait-Si-Ali, S., Akematsu, T., Akira, S., Al-Younes, H. M., Al-Zeer, M. A., Albert, M. L., Albin, R. L., Alegre-Abarrategui, J., Aleo, M. F., Alirezaei, M., Almasan, A., Almonte-Becerril, M., Amano, A., Amaravadi, R. K., Amarnath, S., Amer, A. O., Andrieu-Abadie, N., Anantharam, V., Ann, D. K., Anoopkumar-Dukie, S., Aoki, H., Apostolova, N., Arancia, G., Aris, J. P., Asanuma, K., Asare, N. Y. O., Ashida, H., Askanas, V., Askew, D. S., Auberger, P., Baba, M., Backues, S. K., Baehrecke, E. H., Bahr, B. A., Bai, X.-Y., Bailly, Y., Baiocchi, R., Baldini, G., Balduini, W., Ballabio, A., Bamber, B. A., Bampton, E. T. W., Juhász, G., Bartholomew, C. R., Bassham, D. C., Bast, R. C., Batoko, H., Bay, B.-H., Beau, I., Béchet, D. M., Begley, T. J., Behl, C., Behrends, C., Bekri, S., Bellaire, B., Bendall, L. J., Benetti, L., Berliocchi, L., Bernardi, H., Bernassola, F., Besteiro, S., Bhatia-Kissova, I., Bi, X., Biard-Piechaczyk, M., Blum, J. S., Boise, L. H., Bonaldo, P., Boone, D. L., Bornhauser, B. C., Bortoluci, K. R., Bossis, I., Bost, F., Bourquin, J.-P., Boya, P., Boyer-Guittaut, M., Bozhkov, P. V., Brady, N. R., Brancolini, C., Brech, A., Brenman, J. E., Brennand, A., Bresnick, E. H., Brest, P., Bridges, D., Bristol, M. L., Brookes, P. S., Brown, E. J., Brumell, J. H., Brunetti-Pierri, N., Brunk, U. T., Bulman, D. E., Bultman, S. J., Bultynck, G., Burbulla, L. F., Bursch, W., Butchar, J. P., Buzgariu, W., Bydlowski, S. P., Cadwell, K., Cahová, M., Cai, D., Cai, J., Cai, Q., Calabretta, B., Calvo-Garrido, J., Camougrand, N., Campanella, M., Campos-Salinas, J., Candi, E., Cao, L., Caplan, A. B., Carding, S. R., Cardoso, S. M., Carew, J. S., Carlin, C. R., Carmignac, V., Carneiro, L. A. M., Carra, S., Caruso, R. 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Affiliations
  • University of Michigan (US)
  • Hebrew University of Jerusalem (IL)
  • KU Leuven (BE)
  • Hospital for Sick Children (CA)
  • The University of Tokyo (JP)
  • University of Cincinnati (US)
  • Japan Women's University (JP)
  • University of Massachusetts Chan Medical School (US)
  • University of Connecticut (US)
  • Franche-Comté Électronique Mécanique Thermique et Optique - Sciences et Technologies (FR)
  • University of Pittsburgh (US)
  • Buck Institute for Research on Aging (US)
  • Telethon Institute Of Genetics And Medicine (IT)
  • University of Toledo (US)
  • Iowa State University (US)
  • Centre National de la Recherche Scientifique (FR)
  • Laboratory of Pathogens and Host Immunity (FR)
  • Western University of Health Sciences (US)
  • Indiana University Indianapolis (US)
  • Université Nice Sophia Antipolis (FR)
  • Linköping University (SE)
  • Medical University of Vienna (AT)
  • Institut de Biochimie et Génétique Cellulaires (FR)
  • Instituto de Fermentaciones Industriales (ES)
  • Dulbecco Telethon Institute (IT)
  • Korea Advanced Institute of Science and Technology (KR)
  • The University of Texas Southwestern Medical Center (US)
  • University of Hong Kong (HK)
  • Laboratoire Bordelais de Recherche en Informatique (FR)
  • Peking University (CN)
  • Emory University (US)
  • Brigham and Women's Hospital (US)
  • University of California, Los Angeles (US)
  • University of Oxford (GB)
  • University of Strathclyde (GB)
  • Biology of Infection (FR)
  • Institut de Génomique Fonctionnelle (FR)
  • Institut des Maladies Neurodégénératives (FR)
  • Brunel University of London (GB)
  • University of New Mexico (US)
  • Institut Jacques Monod (FR)
  • Virologie et Pathologies Humaines (FR)
  • National University of General San Martín (AR)
  • Institut de Biologie Intégrative de la Cellule (FR)
  • Institut de Recherche en Infectiologie de Montpellier (FR)
  • University of Brescia (IT)
  • Montreal Neurological Institute and Hospital (CA)
  • University of Rome Tor Vergata (IT)
  • Laboratoire de Biologie du Développement (FR)
  • Microbe, Intestine, Inflammation and Host Susceptibility (FR)
  • Inserm (FR)
  • Institut de Biologie Moléculaire et Cellulaire (FR)
  • Hôpital l'Archet (FR)
  • University of Groningen (NL)
  • National Institute of Advanced Industrial Science and Technology (JP)
  • The University of Texas at San Antonio Health Science Center (US)
  • Hôpital Pasteur (FR)
  • Danish Cancer Society (DK)
  • Institut de Biologie et de Chimie des Protéines (FR)
  • Cancer Research UK (GB)
  • University of Southern California (US)
  • Niigata University (JP)
  • University of Minnesota (US)
  • Wayne State University (US)
  • Weizmann Institute of Science (IL)
  • University Hospitals of Cleveland (US)
  • Christian-Albrechts-Universität zu Kiel (DE)
  • Institut für Immunologie (DE)
  • Centro de Investigacion Principe Felipe (ES)
  • Tokyo Metropolitan Institute of Medical Science (JP)
  • Institut Gustave Roussy (FR)
  • George Washington University (US)
  • Centre for Cancer Biology (AU)
  • University of Chile (CL)
  • Université de Rennes (FR)
  • National Cheng Kung University (TW)
  • Hôpital Paul-Brousse (FR)
  • Howard Hughes Medical Institute (US)
  • National University of Singapore (SG)
  • Institut National de la Recherche Scientifique (CA)
  • Johns Hopkins University (US)
  • University of Graz (AT)
  • Albert Einstein College of Medicine (US)
  • FORTH Institute of Molecular Biology and Biotechnology (GR)
  • Centre Antoine Lacassagne (FR)
  • Vanderbilt University (US)
  • University of Siena (IT)
  • University of Lausanne (CH)
  • Harvard University (US)
  • University of Copenhagen (DK)
  • The University of Texas at San Antonio (US)
  • Institute of Biochemistry (CH)
  • Hôpital Antoine-Béclère (FR)
  • Columbia University (US)
  • University Medical Center Utrecht (NL)
  • University of Cambridge (GB)
  • University of Limerick (IE)
  • Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (FR)
  • Fondazione IRCCS Istituto Nazionale dei Tumori (IT)
  • Hôpital Ophtalmique Jules-Gonin (CH)
  • University of Geneva (CH)
  • Thomas Jefferson University (US)
  • University of Ferrara (IT)
  • Abramson Cancer Center (US)
  • University of Messina (IT)
  • Shandong University (CN)
  • Institute of Modern Physics (CN)
  • Institute of Computing Technology (CN)
  • European Synchrotron Radiation Facility (FR)
  • Leiden University (NL)
  • Institut de Biochimie et Biophysique Moléculaire et Cellulaire (FR)
Corresponding author
Klionsky, Daniel J.
ORCID
Venue
Autophagy
Publisher
Informa UK Limited
Published
2012-04
Volume
8
Issue
4
Pages
445-544
ISSN
  • 1554-8627 (ISSN-L)
  • 1554-8635
DOI
10.4161/auto.19496
PMC
PMC3404883
OpenAlex
W2164778558
Semantic Scholar
e32ad6586854e69af86fd3250f7c3091957fdc3d
Cited by
3,996
References
894
Citations by year
2026: 45 · 2025: 64 · 2024: 98 · 2023: 112 · 2022: 124 · 2021: 245 · 2020: 242 · 2019: 261 · 2018: 261 · 2017: 357 · 2016: 481 · 2015: 599 · 2014: 574 · 2013: 369 · 2012: 105
FWCI
354.83
Type
journal article
Language
en
Open access
Locations
  • Autophagy (publishedVersion)
  • PubMed (publishedVersion)
  • Americanae (AECID Library) (publishedVersion, other-oa)
  • Research Publications (Maastricht University) (publishedVersion, other-oa)
  • Pure Amsterdam UMC (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion)
  • Amsterdam UMC (VU Amsterdam) - Institutional Repository (publishedVersion)
  • SERVAL (Université de Lausanne) (publishedVersion)
  • Archive ouverte UNIGE (University of Geneva) (publishedVersion, other-oa)
  • Repository of the Academy's Library (Library of the Hungarian Academy of Sciences) (acceptedVersion)
  • Sabanci University (acceptedVersion)
  • MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
  • UEA Digital Repository (University of East Anglia) (acceptedVersion)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion)
  • Institutional Research Information System (University of Udine) (submittedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
  • CaltechAUTHORS (California Institute of Technology) (submittedVersion)
  • Bond University Research Portal (Bond University) (submittedVersion)
  • Iowa State University Digital Repository (Iowa State University) (submittedVersion)
  • Rare & Special e-Zone (The Hong Kong University of Science and Technology) (submittedVersion)
  • National University of Singapore (submittedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
  • HAL (Le Centre pour la Communication Scientifique Directe) (submittedVersion)
  • HAL Portal Artxiker (Hindustan Aeronautics Limited (India)) (submittedVersion)
  • Arca - Repositório Institucional da Fiocruz (submittedVersion, other-oa)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion)
  • CINECA IRIS Institutial research information system (University of Pisa) (submittedVersion)
  • Cineca Institutional Research Information System (Tor Vergata University) (submittedVersion)
  • Bern Open Repository and Information System (University of Bern) (submittedVersion)
  • University of Debrecen Electronic Archive (University of Debrecen) (submittedVersion)
  • Digital Access to Libraries (Université catholique de Louvain (UCL), l'Université de Namur (UNamur) and the Université Saint-Louis (USL-B)) (submittedVersion)
  • Consultation of the Doctoral Thesis Database (TESEO) (Ministerio de Educación, Cultura y Deporte) (submittedVersion, cc-by-nc-nd)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion)
  • Adelaide Research & Scholarship (AR&S) (University of Adelaide) (submittedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion)
  • Max Planck Institute for Plasma Physics (submittedVersion)
  • UCL Discovery (University College London) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • Publikationsdatenbank der Fraunhofer-Gesellschaft (Fraunhofer-Gesellschaft) (submittedVersion)
  • University of Debrecen Electronic Archive (University of Debrecen) (submittedVersion, other-oa)
  • ORCA Online Research @Cardiff (Cardiff University) (submittedVersion)
  • The HKU Scholars Hub (University of Hong Kong) (submittedVersion)
  • IrInSubria (University of Insubria) (submittedVersion)
  • Institutional Research Information System (Università degli Studi di Brescia) (submittedVersion)
  • Archive of Doctoral Theses and Digital Collections (University of Calabria) (submittedVersion)
  • IRIS (submittedVersion)
  • IRIS UNIMORE (University of Modena and Reggio Emilia) (submittedVersion, cc-by)
  • Nova Science Publishers (Nova Science Publishers, Inc.) (submittedVersion)
  • IRIS Research product catalog (Sapienza University of Rome) (submittedVersion)
  • Archivio Istituzionale della Ricerca - Università Vita-Salute San Raffaele (submittedVersion)
  • Institutional Research Information System University of Turin (University of Turin) (submittedVersion, other-oa)
  • CNR ExploRA (submittedVersion, other-oa)
  • JuSER (Forschungszentrum Jülich) (submittedVersion)
  • Research Portal (King's College London) (submittedVersion)
  • Lirias (submittedVersion)
  • Lund University Publications (Lund University) (submittedVersion)
  • Leiden Repository (Leiden University) (submittedVersion)
  • CINECA IRIS Institutional Research information system (University of Urbino) (submittedVersion)
  • Open Repository and Bibliography (University of Luxembourg) (submittedVersion)
  • DZNE Pub (submittedVersion)
  • Fraunhofer-Publica (Fraunhofer-Gesellschaft) (submittedVersion)
  • University of Birmingham Research Portal (University of Birmingham) (submittedVersion)
  • Research Explorer (The University of Manchester) (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion, other-oa)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion)
  • Repository of the University of Namur (submittedVersion, other-oa)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, other-oa)
  • Radboud Repository (Radboud University) (submittedVersion)
  • Griffith Research Online (submittedVersion)
  • RVC Research Online (Royal Veterinary College) (submittedVersion, cc-by)
  • CINECA IRIS Institutional Research Information System (University of Bari Aldo Moro) (submittedVersion)
  • SUNScholar (Stellenbosch University) (submittedVersion)
  • Tokyo Tech Research Repository (Tokyo Institute of Technology) (submittedVersion)
  • Archive ouverte UNIGE (University of Geneva) (submittedVersion, cc-by)
  • Use Siena air (University of Siena) (submittedVersion)
  • VinaR (Institute of Nuclear Sciences "Vinča") (submittedVersion, other-oa)
  • VUBIR (Vrije Universiteit Brussel) (submittedVersion)
  • Padua Research Archive (University of Padova) (submittedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion)
  • Research Publications (Maastricht University) (submittedVersion)
  • Research Publications (Maastricht University) (submittedVersion, other-oa)
  • NOVA (University of Newcastle Australia) (submittedVersion)
Funders
  • Howard Hughes Medical Institute
  • U.S. Department of Veterans Affairs (I01 BX001318, I01BX001969)
  • Wellcome Trust (087518, 102974, 095317)
  • Cancer Research UK (11359, 15153, 12103, 15890, 15816, 12825)
  • National Institute for Health and Care Research (NF-SI-0611-10163)
  • Parkinson's UK (G-0905, J-0901)
  • Sight Research UK (pml010004)
  • Brain Tumour Charity
  • Novo Nordisk Fonden (NNF12OC1016434)
  • Versus Arthritis (19379, 17655, 17285)
  • National Institutes of Health (GM53396)
  • Biotechnology and Biological Sciences Research Council (BBS/E/F/00044446, BB/F012861/1, BB/E018521/1, BB/I013695/1, BBS/E/F/00042262, BB/G021422/1, BBS/E/B/00001221)
  • Natural Environment Research Council (pml010004)
  • Japan Society for the Promotion of Science (23617020, 23580099, 24570076, 23790471, 24370077, 22590274, 23689032, 24680038, 23570241)
  • National Institute on Alcohol Abuse and Alcoholism (R37 AA020518, R01 AA019730)
  • National Institute on Aging (R01 AG023039, R01AG039628, R01AG033283, R21 AG039799, R01AG011833, R01 AG031867, K02AG042095, P30AG028740)
  • National Heart, Lung, and Blood Institute (R01HL106019, R01 HL112330, R01 HL079669, R01 HL085629, R01HL060590, R01 HL102738, R01HL067724, R01HL072166, R01HL114858, R01 HL 096625)
  • National Institute of Mental Health (R01 MH073490)
  • National Eye Institute (R01EY020491, R01EY019320, R01 EY009083)
  • National Cancer Institute (R01CA100857, R01CA134530, R01 CA 150214, K05CA-142885, R01CA103632, R01CA123350, R01 CA138641, R01CA129536, R01 CA143811, R01 CA142862, R01CA159314, K08CA164047, R01CA162405, R01 CA137260, R01 CA109182, R01CA139319, R01 CA131188, R01 CA133228, R01CA141703, R01 CA140964, P01 CA108671, R01CA157490, R01CA154835, R01CA102184)
  • National Institute of General Medical Sciences (GM 53396, R01 GM099040, R01 GM062509, R01 GM089919, R01 GM053396, R01GM082830, R01GM097355, R01GM079431, P20GM121176)
  • National Institute of Allergy and Infectious Diseases (P01 AI056097, R37AI042999, R01AI079065, R01 AI089716, R01AI064705, R01AI042999, R01AI080703, R01 AI081884, R01 AI057831)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK082437, R01DK061498, R01 DK079879, R01 DK073336, R01 DK044234, R01 DK050610, R01DK094652, R01 DK100644, R01DK052825, R37DK050107, R01 DK090115, K01 DK087776, R01DK124709, R01DK-074778)
  • National Institute of Neurological Disorders and Stroke (R01NS046451, R01 NS050396, R01-NS064090, R01 NS080108, R01 NS046489, R01NS043466)
  • National Institute of Dental and Craniofacial Research (R01DE010742)
  • National Center for Research Resources (S10 RR025596)
  • National Center for Advancing Translational Sciences (UL1 TR000005)
  • National Institute on Minority Health and Health Disparities (G12 MD007599, G12MD007595, TL4MD009637)
  • National Center for Complementary and Integrative Health (R01 AT005076)
Topics
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
autophagic flux, autophagosomes, autolysosomes, macroautophagy, autophagosome formation, lysosomal degradation, assay standardization, autophagy monitoring
MeSH terms
Animals, Autophagy, Biological Assay, Humans, Models, Biological

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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