Guidelines for the use and interpretation of assays for monitoring autophagy

In the paper

“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”

What the sources say

• ?Not checked — no full text of the sourcecites [419] source
• Disputed

  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.

  cites [421] sourceExperimental — accuracy not yet measured

In the paper

“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”

What the sources say

• Disputed

  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)

  Checked by a reasoning model — may be wrong.

  cites [115] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [435] source

In the paper

“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”

What the sources say

• Disputed

  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.

  cites [497] sourceExperimental — accuracy not yet measured

In the paper

“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”

What the sources say

• Partly supported

  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)

  cites [43] sourceExperimental — accuracy not yet measured

In the paper

“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”

What the sources say

• ?Could not verify

  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)

  cites [23] sourceExperimental — accuracy not yet measured

In the paper

“Autophagosomes (also referred to as initial autophagic vacuoles, AVi) have a double membrane that is usually at least partly visible as two parallel membrane bilayers separated by an electron-lucent cleft (Fig. 2A).45,46”

What the sources say

• Partly supported

  The discussion of Figure 1 (pp. 931–932) says autophagosomes have two limiting membranes made of parallel lipid bilayers, which appear as two dark lines at lower resolution and as layers separated by a light cisternal space at high resolution. Our copy does not identify autophagosomes by the alternate name “initial autophagic vacuoles (AVi).” (LLM, conf=0.85)

  cites [45] sourceExperimental — accuracy not yet measured
• ?Not matched

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

  cites [46]

In the paper

“The maturation from the phagophore through the autolysosome is a dynamic and continuous process, 44 and, thus, the classification of compartments into discrete morphological subsets can be problematic”

What the sources say

• Supported

  In “Identification of autophagic compartments in electron microscopy” (page unavailable), the paper describes phagophores forming autophagosomes, which mature through fusion into amphisomes and autolysosomes. It also describes gradual, overlapping morphological changes during maturation, supporting the paraphrase that the process is dynamic and continuous. (LLM, conf=0.88)

  cites [44] source

In the paper

“Amphisomes 48 can sometimes be identified by the presence of small internal vesicles inside the autophagosome/autophagic vacuole (AV).49”

What the sources say

• ?Not matched

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

  cites [48]
• ?Could not verify

  Figure 8 and the surrounding text (p. 904) describe inclusions inside late autophagic vacuoles, but propose that they arise from secondary membrane invaginations. The paper does not identify these structures as amphisomes or say that small internal vesicles can be used to recognize amphisomes; searches for amphisomes and multivesicular bodies found no passage establishing that criterion. (LLM, conf=0.72)

  cites [49] sourceExperimental — accuracy not yet measured

In the paper

“Autophagosomes contain cytosol and/or organelles that look morphologically intact, i.e., similar to the cytosol and organelles elsewhere in the cell.43,47”

What the sources say

• Supported

  In “The Autophagic Pathways,” the paper says autophagosomes contain cytosol and/or organelles. The same section describes initial autophagic vacuoles, which include autophagosomes, as containing morphologically intact cytosol or organelles. Page numbers are not provided in our copy. (LLM, conf=0.92)

  cites [43] source
• Supported

  In the section on leupeptin-induced alterations, the authors identify autophagosomes by their contents of undegraded cytoplasm (Fig. 6C). They also describe the cytoplasm in related amphisomes as unaltered or only slightly altered, and the introductory passage says autophagy sequesters cytoplasm including organelles. Together, these support the morphological description in the claim. (LLM, conf=0.91)

  cites [47] source

In the paper

“LC3B, or the protein tagged at its N terminus with a fluorescent protein such as GFP (GFP-LC3), has been used to monitor autophagy through indirect immunofluorescence or direct fluorescence microscopy (Fig. 9), measured as an increase in punctate LC3 or GFP-LC3.187,188”

What the sources say

• Partly supported

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

  cites [187] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [188] source

In the paper

“An additional caveat when using the photoactivatable construct PA-GFP 180 is that the process of activation by photons may induce DNA damage, which could, in turn, elicit induction of autophagy.”

What the sources say

• ?Could not verify

  The Results and Discussion passages describe GFP-LC3 fluorescence in lysosomes and the tandem mRFP-GFP-LC3 reporter. Searches for PA-GFP, photoactivation, and light-induced DNA damage found no passage addressing whether activation photons damage DNA or induce autophagy. No page numbers were available in our copy. (LLM, conf=0.72)

  cites [180] sourceExperimental — accuracy not yet measured

In the paper

“Human WIPI1 and WIPI2 function downstream of the phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex I [PIK3C3, BECN1, PIK3R4/ VPS15, ATG14] and upstream of both the ATG12 and LC3 ubiquitin-like conjugation systems.418,420,421”

What the sources say

• ?Not checked — no full text of the sourcecites [418] source
• ?Not checked — no full text of the sourcecites [420] source
• Partly supported

  The Introduction (page unavailable) places WIPI-1 and WIPI-2 downstream of PtdIns(3)P-generating events such as the Beclin 1/PtdIns3KC3 complex. A later passage (page unavailable) places WIPI-1 upstream of both autophagosomal ubiquitin-like conjugation systems, but does not establish that same position for WIPI-2; that part of the claim is unsupported. (LLM, conf=0.85)

  cites [421] sourceExperimental — accuracy not yet measured

In the paper

“Yeast Atg18 412,413 and Atg21 262 (or the mammalian WIPI homologs 414 ) are required for both macroautophagy (i.e., nonspecific sequestration of cytoplasm) and autophagy-related processes (e.g., the Cvt pathway, 415,416 specific organelle degradation, 74 and autophagic elimination of invasive microbes 77,78,80,81,417 ).412”

What the sources say

• ?Not checked — no full text of the sourcecites [412] source
• Partly supported

  The Introduction and Discussion report that yeast Aut10p is required for both autophagy and the Cvt pathway. Our copy does not identify Aut10p as Atg18 or establish requirements for Atg21, mammalian WIPI homologs, organelle degradation, or elimination of invasive microbes; those extensions are not supported by this source. (LLM, conf=0.78)

  cites [413] sourceExperimental — accuracy not yet measured
• ?Could not verify

  The Results and Discussion passages address EGFP’s effects on polyubiquitination, not the roles of Atg18, Atg21, or WIPI homologs. Searches for those proteins and for macroautophagy, the Cvt pathway, and autophagic degradation found no passage in our copy addressing the claim. (LLM, conf=0.78)

  cites [262] sourceExperimental — accuracy not yet measured

In the paper

“BECN1/Atg6 and PIK3C3/VPS34 are essential partners in the autophagy interactome that signals the onset of autophagy, 404,423,424”

What the sources say

• Partly supported

  The Results report that yeast Vps30p/Apg6p and Vps34p interact, and the Discussion says both are components of a complex required for autophagy. The paper studies yeast proteins; it does not establish a finding about human BECN1/PIK3C3 or show that their complex signals the onset of autophagy. (LLM, conf=0.85)

  cites [404] sourceExperimental — accuracy not yet measured
• Supported

  The 'Class III phosphatidylinositol 3-kinase complex' section identifies Beclin 1 (the Atg6 homolog) and hVps34 as members of a conserved mammalian complex. It also says the corresponding yeast complex is required for autophagy induction and describes a role for the mammalian complex in initiating recruitment of autophagy machinery. The supplied passages do not give page numbers. (LLM, conf=0.90)

  cites [423] source
• ?Not checked — no full text of the sourcecites [424] source

In the paper

“TORC1 is downstream of AKT1; however, oxidative stress inhibits MTOR, thus allowing autophagy, despite the concomitant activation of AKT1.115”

What the sources say

• ?Could not verify

  The Discussion passage describes PINK1’s involvement in the Beclin1–Vps34 autophagy pathway, but does not address whether TORC1 is downstream of AKT1. Searches for that relationship and for the sentence’s oxidative-stress/MTOR context found no passage in our copy that establishes it; no page number was provided. (LLM, conf=0.74)

  cites [115] sourceExperimental — accuracy not yet measured

In the paper

“Yeast Atg9 may cycle between the phagophore assembly site (PAS) and peripheral reservoirs; 397 the latter correspond to tubulovesicular clusters that are precursors to the phagophore.398”

What the sources say

• Supported

  Citation 397 is attached to the first clause, about Atg9 cycling between the PAS and peripheral sites. The Introduction says Atg9 cycles between the PAS and punctate structures dispersed in the cytosol, and the results identify a mitochondrial pool. The tubulovesicular-cluster description is attached to citation 398, not this source. (LLM, conf=0.91)

  cites [397] source
• Supported

  The Introduction states that yeast Atg9 cycles between peripheral reservoirs and the PAS, and describes the reservoirs as clusters of vesicles and tubules that give rise to the PAS and phagophore. The Discussion likewise proposes that a reservoir acts as a pre-PAS. Page numbers are not available in our copy. (LLM, conf=0.93)

  cites [398] source

In the paper

“The C-terminal fragment of the protein is named the BATS domain (BARKOR/ATG14(L) autophagosome targeting sequence), and is able to direct GFP and BECN1 to autophagosomes in the context of a chimeric protein.409”

What the sources say

• Supported

  The Results identify BATS as the final 80 amino acids of Barkor/Atg14(L) and show that a Beclin 1–BATS chimera localizes to LC3-positive autophagosomes. The Results also report that GFP-tagged BATS marks LC3-positive autophagic structures. Page numbers are not available in our copy. (LLM, conf=0.92)

  cites [409] source

In the paper

“Retrograde movement requires Atg1-Atg13, Atg2-Atg18 and the PtdIns3K complex I. 399”

What the sources say

• Supported

  The Results state that Atg9 cycling through the pre-autophagosomal structure depends on the Atg1–Atg13 complex and that correct Atg9 trafficking additionally requires Atg2, Atg18, and PtdIns 3-kinase complex I. The Discussion identifies this trafficking as retrieval, or retrograde transport, from that structure. (LLM, conf=0.94)

  cites [399] source

In the paper

“Also, persistent autophagy induction can cause negative feedback that results in the reactivation of MTOR under conditions of ongoing starvation.389”

What the sources say

• Supported

  The indexed Abstract states that mTOR is reactivated with prolonged starvation and that this reactivation is autophagy-dependent. It also describes a negative-feedback mechanism in which products of autophagic degradation appear to trigger mTOR reactivation, which then inhibits autophagy. (LLM, conf=0.94)

  cites [389] source

In the paper

“In this regard, the transcriptional induction of LC3 may be necessary to replenish the LC3 protein that is turned over during extensive ER stress-and hypoxia-induced autophagy.443,448”

What the sources say

• ?Not checked — no full text of the sourcecites [443] source
• ?Could not verify

  The available indexed passages are bibliographic material, not the paper’s discussion or results. Searches for LC3 replenishment, protein turnover, and transcriptional induction during ER stress or hypoxia returned no passage addressing the proposed mechanism, so our copy does not establish it. (LLM, conf=0.70)

  cites [448] sourceExperimental — accuracy not yet measured

In the paper

“The induction of autophagy in certain scenarios is accompanied by an increase in the mRNA levels of certain autophagy genes, such as ATG8/ Lc3 441,442 and Atg12, 443”

What the sources say

• Supported

  In the section on autophagic degradation in HeLa cells, the paper reports that LC3 mRNA increased during amino-acid starvation and links this to induction of autophagy. This supports the LC3/ATG8 portion of the sentence, to which the citation is attached; Atg12 has a separate citation. (LLM, conf=0.92)

  cites [441] source
• Supported

  The Results (Expression of APG8) report that yeast APG8 mRNA increased drastically in response to starvation, a condition under which autophagy is induced. This supports the ATG8 portion attributed to this source; the citing sentence assigns Atg12 to a separate citation. (LLM, conf=0.92)

  cites [442] source
• ?Not checked — no full text of the sourcecites [443] source

In the paper

“The ER population of ZFYVE1 is involved in formation of the omegasome.437”

What the sources say

• Partly supported

  The Results (‘Interaction of omegasomes with autophagosomes and the ER’; page not provided) show ER-associated DFCP1 concentrating and expanding into an omegasome, supporting the connection between the ER population and omegasome formation. They do not establish that DFCP1 has a functional role in forming omegasomes; the Results (‘Down-regulation of DFCP1 does not inhibit autophagy’) report that reducing DFCP1 did not inhibit the measured autophagy response. (LLM, conf=0.84)

  cites [437] sourceExperimental — accuracy not yet measured

In the paper

“The relationship between BECN1 and BCL2 is more complex in developing cerebellar neurons as it appears that the cellular levels of BCL2 are, in turn, post-translationally regulated by an autophagic mechanism linked to a switch from immaturity to maturity.429,430”

What the sources say

• ?Not checked — no full text of the sourcecites [429] source
• Partly supported

  The article title states that autophagy regulates post-translational cleavage of BCL-2, and the keywords identify cerebellar granule cells and post-natal development. The available passages do not establish regulation of cellular BCL-2 levels specifically, a link to the immaturity-to-maturity switch, or the claimed BECN1–BCL-2 relationship. (LLM, conf=0.76)

  cites [430] sourceExperimental — accuracy not yet measured

In the paper

“The time course of autophagy as a consequence of DRAM1 activation can be monitored following the disappearance of the VRK1 protein, a direct target of this process, by immunoblot.439”

What the sources say

• Supported

  The Results section on VRK1 downregulation after DNA damage reports a time-course assay in WS1 cells: DRAM accumulation was followed by a reduction in VRK1, quantified from a blot (Fig. 4A; page unavailable). The Results section on DRAM and Beclin-1 knockdown links that VRK1 degradation to the autophagic pathway and shows VRK1 levels measured by blot at different time points (Fig. 5A; page unavailable). Here, VRK1 is a target for degradation, not a protein shown to bind DRAM directly. (LLM, conf=0.90)

  cites [439] source

In the paper

“In addition to the ATG genes, transcriptional upregulation of VMP1 (a protein that is involved in autophagy regulation and that remains associated with the completed autophagosome) can be detected in mammalian cells subjected to rapamycin treatment or starvation, and in tissues undergoing disease-induced autophagy.461”

What the sources say

• Partly supported

  The Results report increased VMP1 mRNA in starved HeLa cells and increased VMP1 mRNA and protein after rapamycin treatment. In pancreas tissue undergoing pancreatitis-induced autophagy, the Results show increased VMP1 protein, but do not establish transcriptional upregulation there. The paper also places VMP1 in autophagic vacuole membranes without establishing that it remains associated with completed autophagosomes. (LLM, conf=0.86)

  cites [461] sourceExperimental — accuracy not yet measured

In the paper

“Transfection of lymphoblastoid cells with a plasmid encoding NeoR-GFP followed by incubation in the presence of 3-MA leads to an accumulation of the NeoR-GFP protein as measured by flow cytometry.488”

What the sources say

• Supported

  The Autophagy inhibitor experiments section describes introducing a NeoR-GFP-expressing plasmid into LCL cells and measuring protein levels by flow cytometry after 3-MA exposure. The CD4+ T cell recognition section reports that 3-MA treatment caused NeoR-GFP to accumulate relative to untreated cells, by about 2.5-fold after one day and 3.5-fold after four days at 10 mM. (LLM, conf=0.95)

  cites [488] source

In the paper

“For example, the neomycin phosphotransferase II-GFP (NeoR-GFP) fusion protein is a target of autophagy.487”

What the sources say

• Supported

  Section 2.5 tests autophagic transport using cells expressing NeoR-GFP and reports increased fluorescence when autophagy is inhibited with 3-MA. The Fig. 5 caption identifies autophagy as the route by which NeoR reaches the endosomal/lysosomal compartment. (LLM, conf=0.93)

  cites [487] source

In the paper

“PHO8 encodes the vacuolar alkaline phosphatase, which is synthesized as a zymogen before finally being transported to and activated in the vacuole.489”

What the sources say

• Supported

  The Introduction identifies ALP as the PHO8 gene product and says it is synthesized as a zymogen. The discussion under “Vacuolar sorting of ALP in vps mutants” states that the inactive precursor is delivered to the vacuole, where removal of a C-terminal propeptide activates it. (LLM, conf=0.94)

  cites [489] source

In the paper

“The precursor form of aminopeptidase I (prApe1) is the major cargo of the Cvt pathway in yeast, a biosynthetic autophagy-related pathway.82”

What the sources say

• ?Could not verify

  The Introduction discusses macroautophagy, and the results examine Listeria infection in macrophages, but neither addresses the Cvt pathway or its cargo. Searches of our full-text copy for prApe1, precursor aminopeptidase I, and the yeast Cvt pathway found no passage supporting the claim; page numbers were unavailable. (LLM, conf=0.75)

  cites [82] sourceExperimental — accuracy not yet measured

In the paper

“The 44-kDa full-length BHMT protein is cleaved in hepatocyte amphisomes in the presence of leupeptin to generate 32-kDa and 10-kDa fragments.[482][483][484][485]”

What the sources say

• ?Not checked — no full text of the sourcecites [482] source
• Partly supported

  The Discussion identifies intact BHMT as p44 and reports that p32 accumulates as its degradation product in autolysosomes in the presence of leupeptin/E64c. The paper does not report a 10-kDa fragment or locate this cleavage in hepatocyte amphisomes; it describes autophagosomes maturing into autolysosomes instead. (LLM, conf=0.90)

  cites [483] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [484] source

In the paper

“A modified version of this marker, GST-BHMT, can be expressed in other cell lines where it behaves similar to the wild-type protein.486”

What the sources say

• Partly supported

  The paper’s available research passage reports that GST-BHMT shows starvation-induced fragmentation in a variety of cell lines. It does not compare that behavior with wild-type BHMT, so the claimed similarity is unsupported by our copy. (LLM, conf=0.82)

  cites [486] sourceExperimental — accuracy not yet measured

In the paper

“For example, mitoPho8D60 can be used to quantitatively measure mitophagy.498”

What the sources say

• Supported

  The Discussion (“Different Methods to Monitor Mitophagy”) explicitly states that the mitoPho8Δ60 assay measures mitophagy quantitatively. The Results describe measuring its alkaline phosphatase activity to assess mitophagy. (LLM, conf=0.95)

  cites [498] source

In the paper

“In addition, mitophagy can also be induced by culturing the cells in a nonfermentable carbon source to postlog phase.”

What the sources say

• ?Could not verify

  The Intracellular Trafficking section discusses autophagy of cellular material, including mitochondria, in endothelial cells, but does not describe inducing mitophagy by culturing cells on a nonfermentable carbon source to postlog phase. Searches for that condition and for yeast growth found no supporting passage in our copy. (LLM, conf=0.76)

  cites [75] sourceExperimental — accuracy not yet measured
• Supported

  The paper’s “Induction and regulation of mitophagy in yeast” section says mitophagy is induced when yeast grown with a non-fermentable carbon source reaches stationary phase. Its “Summary of mitophagy” repeats this point, supporting the citing sentence’s reference to postlog phase. Page numbers were not supplied in the passages. (LLM, conf=0.93)

  cites [516] source
• ?Not checked — no full text of the sourcecites [517] source

In the paper

“Mitophagy can also be quantitatively monitored using a mitochondria-targeted version of the pHdependent Keima protein.549”

What the sources say

• Supported

  The section “Quantitative Observation of the Process of a Single Autophagic Event” states that the authors targeted pH-sensitive mKeima to mitochondria to quantitatively monitor mitophagy. The “Visualizing Parkin-Dependent Delivery of Impaired Mitochondria to Lysosomes” section describes the mitochondria-targeted probe and its pH-dependent imaging readout. (LLM, conf=0.95)

  cites [549] source

In the paper

“Following glucose replenishment after 3 d glucose starvation, the gluconeogenic enzymes are delivered to the vacuole for degradation.538”

What the sources say

• Partly supported

  The opening summary and Results (page unavailable in our copy) report that glucose replenishment targets the gluconeogenic enzyme FBPase to the vacuole for degradation. The paper does not establish this for gluconeogenic enzymes generally or specify the claimed three days of glucose starvation; its Experimental Procedures describe other growth conditions and durations. (LLM, conf=0.86)

  cites [538] sourceExperimental — accuracy not yet measured

In the paper

“Along these lines, it should also be realized that selective types of autophagy continuously occur at a low level under noninducing conditions.”

What the sources say

• Partly supported

  The Discussion says that peroxisomes, or a subset of them, are constitutively degraded by autophagy during normal vegetative growth, without nitrogen depletion or a change in carbon source. This supports continuous selective autophagy under noninducing conditions in H. polymorpha, but the paper does not establish that selective types generally occur at a low level. (LLM, conf=0.84)

  cites [519] sourceExperimental — accuracy not yet measured

In the paper

“It should be noted that fluorescence microscopy has also been used to monitor the half-life of autophagosomes, monitoring GFP-LC3 in the presence and absence of bafilomycin A 1 or following GFP-LC3 after starvation and recovery in amino acid-rich medium”

What the sources say

• ?Not checked — no full text of the sourcecites [18] source
• Partly supported

  The ‘Monitoring Flux Through the Autophagy Pathway’ section describes fluorescence-based tracking of LC3 after starvation and return to rich medium, and compares GFP-LC3-positive vacuoles with and without bafilomycin A1. It measures autophagosome turnover in primary mouse macrophages, but does not report an autophagosome half-life. (LLM, conf=0.82)

  cites [605] sourceExperimental — accuracy not yet measured

In the paper

“For example, variations in basal flux in the liver associated with circadian rhythm may be several fold, which can equal or exceed the changes due to starvation.”

What the sources say

• Partly supported

  The ‘Autophagy undergoes diurnal rhythm during the day’ section reports that liver autophagy flux peaks during the circadian cycle and is modest during the dark phase. Neither that section nor the Discussion quantifies the variation as several-fold or shows that it equals or exceeds the change caused by starvation; the starvation passage concerns gene expression rather than a comparison of flux. (LLM, conf=0.86)

  cites [467] sourceExperimental — accuracy not yet measured

In the paper

“A special caution should also be taken when describing developmental programmed cell death in plants, which is in most cases executed by the growing lytic vacuoles and therefore is referred to as “vacuolar cell death.”671”

What the sources say

• Supported

  The Abstract (page not provided) says vacuolar cell death is common during tissue and organ formation and elimination. The section “Definition of ‘Vacuolar’ Plant Cell Death” describes expanding lytic vacuoles, their role in dismantling the cell, and the name the authors recommend for this form of death. Together, these support the citing sentence’s paraphrase. (LLM, conf=0.89)

  cites [671] source

In the paper

“Furthermore, many of the papers claiming a causative role of autophagy in cell death fail to provide adequate evidence.666”

What the sources say

• Supported

  The “Autophagic Cell Death” section says that signs of autophagy in dying cells have been mistaken for evidence that autophagy caused death, and that some resulting claims were possibly artefactual. The paper also says that establishing autophagic cell death requires showing that inhibiting autophagy prevents death. Together, these passages support the citing sentence’s broad criticism of the evidence behind claims of a causative role. (LLM, conf=0.86)

  cites [666] source

In the paper

“MTORindependent regulation can be achieved through lithium, sodium valproate and carbamazepine, compounds that lower the myoinositol-1,4,5-triphosphate levels.765”

What the sources say

• Partly supported

  The Results section on mTOR inhibition reports that lithium induces autophagy without reducing markers of mTOR activity, and the Results section on IMPase reports that lithium lowers IP3. The section on mood-stabilizing drugs reports inositol depletion and enhanced clearance of mutant proteins with carbamazepine, with similar valproate results described as unpublished. It does not establish that carbamazepine or valproate lowered IP3 or regulated autophagy independently of mTOR. (LLM, conf=0.85)

  cites [765] sourceExperimental — accuracy not yet measured

In the paper

“Currently, the most commonly used inducer of autophagy is rapamycin, an allosteric inhibitor of TORC1”

What the sources say

• Partly supported

  The Introduction calls rapamycin a specific inhibitor of Tor, supporting the inhibition part of the claim. Our copy does not identify the target as TORC1 or describe the inhibition as allosteric (searched: “allosteric inhibitor TORC1” and “rapamycin TOR complex TORC1 FKBP12 binding mechanism”). (LLM, conf=0.88)

  cites [379] sourceExperimental — accuracy not yet measured
• ?Not matched

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

  cites [762][763][764]

In the paper

“Currently, the most commonly used inducer of autophagy is rapamycin”

What the sources say

• Partly supported

  The Introduction states that rapamycin induces autophagy in yeast, and the Results describe effects of rapamycin treatment. Neither passage establishes that it is the most commonly used inducer of autophagy; the claim's comparative usage assertion is unsupported. (LLM, conf=0.84)

  cites [379] sourceExperimental — accuracy not yet measured
• ?Not matched

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

  cites [762][763][764]

In the paper

“In the methylotrophic yeast species Pichia pastoris and Hansenula polymorpha a shift of cells grown in batch from glucose to methanol results in stimulation of autophagy.776,777”

What the sources say

• ?Not checked — no full text of the sourcecites [776] source
• Supported

  The Methods (Organisms & Growth; page unavailable) state that H. polymorpha was grown in batch cultures on glucose before transfer to methanol medium. The Results and discussion (Autophagy; page unavailable) report strong morphological evidence of increased autophagy after the shift. This paper supports the H. polymorpha part of the two-species sentence; the P. pastoris part is also accompanied by another citation. (LLM, conf=0.92)

  cites [777] source

In the paper

“Along these lines, the use of whole embryos makes it possible to investigate autophagy in multipotent cells, which interact among themselves in their natural environment, bypassing the disadvantages of isolated cells that are deprived of their normal network of interactions.632”

What the sources say

• Supported

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

  cites [632] source

In the paper

“but ammomium chloride (or chloroquine) may be more effective.”

In the paper

“The saturating concentrations of these lysosomal inhibitors vary in different cell lines”

In the paper

“Under certain conditions [such as Earle's balanced salt solution (EBSS)induced starvation] in some cell lines, when the lysosomal pH becomes particularly low, free GFP is undetectable because both the LC3-II and free GFP fragments are quickly degraded.172”

What the sources say

• ?Not checked — no full text of the sourcecites [172] source

In the paper

“The phagophore and the limiting membranes of autophagosomes contain few, or no detectable, IMPs (Fig. 3A and B), when compared with other cellular membranes and to the membranes of lysosomes.”

In the paper

“whereas a 100 mM concentration with 4 h pre-incubation had a partial effect.”

In the paper

“10 mM NH 4 Cl (or 30 mM chloroquine, not shown) were the most effective compounds for demonstrating the accumulation of LC3-II.”

In the paper

“Pepstatin A at 10 mg/ml with a 1 h pre-incubation was not effective at blocking degradation”

In the paper

“The main limitation of the GFP-LC3 processing assay in mammalian cells is that it seems to depend on cell type and culture conditions (Hosokawa N, Mizushima N, unpublished data).”

What the sources say

• ?Not matched

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

  cites Hosokawa N, Mizushima N, unpublished data

In the paper

“It is possible to circumvent this latter problem by imaging paraformaldehyde-fixed cultures that are maintained in a neutral pH buffer, which retains EGFP fluorescence (Kleinman M, Reiners JJ, personal communication).”

What the sources say

• ?Not matched

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

  cites Kleinman M, Reiners JJ, personal communication

In the paper

“free GFP can be detected when cells are treated with nonsaturating doses of lysosomal inihibitors such as chloroquine, E-64d and bafilomycin A 1 .”

In the paper

“E-64d was also effective in preventing the degradation of LC3-II, with or without a preincubation”

In the paper

“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);”

In the paper

“we do not know, at present, the actual mechanistic relationship between Atg8-PE/LC3-II formation and the rest of the autophagic process”

In the paper

“TEM can be used to monitor both selective and nonselective autophagy.”

In the paper

“whereas decreased flux could also be due to inefficient degradation of the cargo once fusion has occurred.19”

What the sources say

• ?Not checked — no full text of the sourcecites [19] source

In the paper

“whereas an increase in autolysosomes may reflect a reduction in degradative activity.”

In the paper

“Importantly, there are no absolute criteria for determining autophagic status that are applicable in every biological or experimental context.”

In the paper

“This is because some assays are inappropriate, problematic or may not work at all in particular cells, tissues or organisms.[3][4][5][6]”

What the sources say

• ?Not matched

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

  cites [3][4][5][6]

In the paper

“The key issue is to differentiate between the often transient accumulation of autophagosomes due to increased induction, from accumulation due to inefficient completion of autophagy, by measuring both the levels of autophagosomes at static time points and by addressing changes in the rates of autophagic degradation of cellular components.19”

What the sources say

• ?Not checked — no full text of the sourcecites [19] source

In the paper

“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 the paper

“and, thus, the classification of compartments into discrete morphological subsets can be problematic; however, some basic guidelines can be offered.”

In the paper

“Furthermore, the use of multiple time points and/or lysosomal fusion/degradation inhibitors can turn even a typically static method such as TEM into one that monitors flux.”

In the paper

“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.”

In the paper

“This secondary autophagic response can complicate the analysis of the autophagy flux, making it appear more vigorous than it would in the absence of the lysosomal inhibitors.”

In the paper

“It should be emphasized that not all vesicles containing electrondense amorphous material are autolysosomes.”

In the paper

“and pepstatin A must be used at saturating conditions to have any noticeable effect.”

In the paper

“For many biological and pathological situations, examination of both early and late autophagic structures yields valuable data regarding the overall autophagy/ lysosomal status in the cells.17”

What the sources say

• ?Not checked — no full text of the sourcecites [17] source

In the paper

“Atg4 cleaves the residue(s) that follow the C-terminal glycine of Atg8/LC3 that will be conjugated to PE.”

In the paper

“Autolysosomes are generally delimited by a single membrane because, in addition to the engulfed material, the inner membrane is degraded by the lytic enzymes.”

In the paper

“the absence of an effect on the total number of GFP-LC3 puncta or on the percentage of cells displaying numerous puncta is indicative of a defect(s) in autophagic flux.”

What the sources say

• ?Not checked — no full text of the sourcecites [177] source

In the paper

“The detection of GFP-LC3/Atg8 is also useful for in vivo studies using transgenic organisms such as Caenorhabditis elegans, 189 Dictyostelium discoideum, 190 filamentous ascomycetes, [191][192][193][194][195] Ciona intestinalis, 196 Drosophila melanogaster, [197][198][199] Arabidopsis thaliana, 200 Leishmania major 202,203 and mice.126”

What the sources say

• ?Not checked — no full text of the sourcecites [189] source
• ?Not checked — no full text of the sourcecites [190] source
• ?Not checked — no full text of the sourcecites [126] source

In the paper

“conversely a decline implies that GFP-LC3 is consumed within newly formed autolysosomes.”

In the paper

“because the stability of GFP is affected by lysosomal pH”

In the paper

“The combination of protease inhibitors (to prevent the degradation of GFP) or compounds that modify lysosomal pH such as NH 4 Cl or chloroquine, or compounds such as bafilomycin A 1 or others that block fusion of autophagosomes with lysosomes (e.g., vinblastine) may be most effective in preventing lysosome-dependent decreases in GFP-LC3 puncta.”

In the paper

“A potential solution to this problem for following fluorescence is to use a photoactivatable version of the fluorescent protein, 176 which allows this assay to be performed essentially as a pulse/chase analysis.”

What the sources say

• ?Not checked — no full text of the sourcecites [176] source

In the paper

“In some autophagyinducing conditions it is possible to observe multi-lamellar membrane structures in addition to the conventional doublemembrane autophagosomes.”

In the paper

“the presence of lysosomal inhibitors should increase the number of GFP-LC3-positive structures”

In the paper

“In subsequent stages of the autophagic process the fusion of the autophagosome with an endosome and a lysosome results in increased density of IMPs in the membrane of the formed autophagic compartments (amphisomes, autolysosomes; Fig. 3C).8,24,[53][54][55][56]61,62”

What the sources say

• ?Not matched

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

  cites 8,24,[53][54][55][56]61,62

In the paper

“red fluorescent proteins (even the monomeric forms) can be toxic due to aggregation.183”

What the sources say

• ?Not checked — no full text of the sourcecites [183] source

In the paper

“Thus, alkalinizing compounds are more effective in blocking LC3-II degradation”

In the paper

“a constant increase in the number of cells accumulating GFP-LC3 puncta is suggestive of defective fusion of autophagosomes”

In the paper

“TEM observations of platinum-carbon replicas obtained by the freeze fracture technique can also supply useful ultrastructural information on the autophagic process.”

In the paper

“it can be problematic to use GFP fluorescence to follow flux, as new GFP-LC3 is continuously being synthesized.”

In the paper

“caution is required when evaluating the localization of mCherry fusion proteins during autophagy due to the persistence of the mCherry signal in acidic environments”

In the paper

“it may be preferable to use a different fluorophore such as monomeric red fluorescent protein (mRFP) or mCherry, which retain fluorescence even at acidic pH.181”

What the sources say

• ?Not checked — no full text of the sourcecites [181] source

In the paper

“Accordingly, it may be possible to follow the formation and subsequent disappearance of WIPI puncta to monitor autophagy induction and flux using specific techniques.”

In the paper

“with stable transformants more cells can be easily analyzed because nearly 100% of the population will express tagged LC3.”

In the paper

“The use of imaging cytometry allows rapid and quantitative measures of the number of LC3 puncta and their relative number in individual or mixed cell types, using computerized assessment, enumeration, and data display (e.g., see refs.142 and 210).”

What the sources say

• ?Not checked — no full text of the sourcecites [142] source
• ?Not checked — no full text of the sourcecites [210] source

In the paper

“On the basis of quantitative fluorescence microscopy, this specific WIPI protein localization has been used as an assay to monitor autophagy in human cells.419”

What the sources say

• ?Not checked — no full text of the sourcecites [419] source

In the paper

“Accordingly, WIPI puncta can be assessed as an alternative to LC3.”

In the paper

“One final parameter that may be considered is that the total amount of the ATG12-ATG5 conjugate may increase following prolonged starvation as has been observed in hepatocytes and both mouse and human fibroblasts (Cuervo AM, personal communication; Sarkar S, personal communication).”

What the sources say

• ?Not matched

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

  cites Cuervo AM, personal communication
• ?Not matched

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

  cites Sarkar S, personal communication

In the paper

“Therefore, monitoring ATG12-ATG5 conjugation per se may not be a useful method for following the induction of autophagy.”

In the paper

“Thus, assessing the mRNA levels of LC3 and other autophagy-related genes by northern blot or qRT-PCR may provide correlative data relating to the induction of autophagy.”

In the paper

“When a BECN1-independent autophagy pathway is induced, such aggregates are not formed regardless of the fact that the cell expresses BECN1 (e.g., as assessed by western blotting) (Isidoro C, personal communication).”

What the sources say

• ?Not matched

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

  cites Isidoro C, personal communication

In the paper

“Similarly, a stimulus-dependent increase in LC3B expression is detected in neutrophils undergoing autophagy induction.445”

What the sources say

• ?Not checked — no full text of the sourcecites [445] source

In the paper

“BECN1 is inhibited by its binding to the anti-apoptotic protein BCL2.425”

What the sources say

• ?Not checked — no full text of the sourcecites [425] source

In the paper

“Conclusion: Measuring the turnover of long-lived proteins is a good method for determining autophagic flux.”

In the paper

“This means that the results regarding specific changes in autophagic cells could be hidden due to the background of the average data.”

In the paper

“In some cases (e.g., yeast ATG14), increased transcription is not accompanied by increased protein levels, apparently due to changes in translation efficiency under starvation conditions (Abeliovich H, unpublished data).”

What the sources say

• ?Not matched

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

  cites Abeliovich H, unpublished data

In the paper

“Finally, inhibition of a particular degradative pathway is typically accompanied by an increase in a separate pathway as the cell attempts to compensate for the loss of degradative capacity.158,478,493”

What the sources say

• ?Not checked — no full text of the sourcecites [158] source
• ?Not checked — no full text of the sourcecites [478] source
• ?Not checked — no full text of the sourcecites [493] source

In the paper

“whereas mitophagy can be monitored by the generation of free GFP from Om45-GFP, Idh1-GFP, Idp1-GFP or mito-DHFR-GFP.[496][497][498][499][500]”

What the sources say

• ?Not matched

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

  cites [496][497][498][499][500]

In the paper

“The resulting activation of the zymogen can be easily measured by assays for alkaline phosphatase.178”

What the sources say

• ?Not checked — no full text of the sourcecites [178] source

In the paper

“It is also possible to monitor pexophagy in yeasts by the disappearance of activities of specific peroxisome markers such as catalase, alcohol oxidase or amine oxidase in cell-free extracts, 504 or permeabilized cell suspensions.”

What the sources say

• ?Not checked — no full text of the sourcecites [504] source

In the paper

“Catalase activity, however, is a useful marker only when peroxisomal catalases are the only such enzymes present.”

In the paper

“It should be noted that this method monitors bulk autophagy since it relies on the passive transit of a cytoplasmic marker to the lysosome.”

In the paper

“It should be noted that in addition to carbon source change, simultaneous nitrogen starvation is also required for efficient mitophagy induction.”

In the paper

“The relative resistance or sensitivity to an exogenous protease in the absence of detergent is an indication of whether the autophagosome (or other sequestering vesicle) is complete or incomplete, respectively.”

In the paper

“However, staining with certain MitoTracker dyes depends on mitochondrial activity and membrane potential, so that damaged, or sequestered nonfunctional mitochondria may not be stained.”

In the paper

“The Cvt complex (prApe1 bound to Atg19) is smaller than typical peroxisomes or mitochondrial fragments that are subject to autophagic degradation.”

In the paper

“First, as discussed in Selective types of autophagy, prApe1 is not an accurate marker for nonspecific autophagy; import of prApe1 utilizes a receptor (Atg19) and a scaffold (Atg11) that make the process specific.”

In the paper

“These assays can also be modified to measure autophagic flux.”

In the paper

“In addition, vesicles that are substantially smaller than autophagosomes can effectively sequester the Cvt complex.”

In the paper

“This process can be monitored by western blot, following the generation of free GFP from Rpl25-GFP or the disappearance of ribosomal subunits such as Rps3.”

In the paper

“Sequestration assays present another valid method for monitoring autophagy, and in particular for discriminating between conditions where the autophagosome is complete (but not fused with the lysosome/vacuole) or open (i.e., a phagophore).”

In the paper

“At present, the prApe1 assay cannot be used in any system other than yeast.”

In the paper

“In addition, the sequestration of prApe1 is specific, even under starvation conditions, as it involves the recognition of the propeptide by a receptor, Atg19, which in turn interacts with the scaffold protein Atg11.530,531”

What the sources say

• ?Not checked — no full text of the sourcecites [530] source
• ?Not checked — no full text of the sourcecites [531] source

In the paper

“Accordingly, PMN can be monitored using the proteins that are associated with the NVJs as markers.”

In the paper

“The turnover of the autophagosome or the autolysosome will be differentially affected if fusion or intralysosomal degradation is inhibited.14,16,24,604”

What the sources say

• ?Not checked — no full text of the sourcecites [14] source
• ?Not checked — no full text of the sourcecites [16] source
• ?Not checked — no full text of the sourcecites [24] source

In the paper

“Activation of the UPR in the ER in yeast induces a type of selective macroautophagy of the ER.[525][526][527]”

What the sources say

• ?Not matched

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

  cites [525][526][527]

In the paper

“In particular, it is not sufficient to follow a single mitochondrial outer membrane protein because these can be degraded independently of mitophagy.”

In the paper

“A similar carbon source change from oleic acid or methanol to ethanol or glucose (with or without nitrogen starvation) can be used to assay for pexophagy.515”

What the sources say

• ?Not checked — no full text of the sourcecites [515] source

In the paper

“To quantitatively follow PMN, an assay analogous to the above-described GFP-Atg8/LC3 processing assay has been established using either GFP-Osh1 or Nvj1-GFP.”

In the paper

“For example, to induce a substantial level of mitophagy, cells need to be precultured in a nonfermentable carbon source such as lactate or glycerol to stimulate the proliferation of mitochondria.”

In the paper

“Thus, this method also distinguishes between a block in autophagosome formation vs. fusion with the vacuole.”

In the paper

“Some experiments require the use of inhibitors (e.g., 3-MA or wortmannin) or overexpression of proteins (e.g., RAB7 dominant negative mutants) that may also affect the endocytic pathway or the delivery of DQ-BSA to lysosomes (e.g., wortmannin causes the swelling of late endosomes 611 ).”

What the sources say

• ?Not checked — no full text of the sourcecites [611] source

In the paper

“Controlling for circadian effects can greatly reduce the mouse-tomouse variability in autophagy markers and flux (Haspel JA, Choi AMK, personal communication).”

In the paper

“Considering the limited methods available for in vivo analysis of autophagy, tissue fractionation is a valid, although relatively laborious, method for monitoring this process.”

In the paper

“Thus, DQ-BSA is useful for detecting intracellular proteolytic activity as a measure of a functional lysosome.610”

What the sources say

• ?Not checked — no full text of the sourcecites [610] source

In the paper

“However, the ubiquitin-proteasome system likely confounds this investigation, as this pathway can also be inhibited by insulin and amino acids.653”

What the sources say

• ?Not checked — no full text of the sourcecites [653] source

In the paper

“In these settings, GFP fluorescent puncta are indicative of autophagic structures.”

In the paper

“Along these lines, autophagic flux can be determined by western blot in retinas placed in culture for 4 h with protease inhibitors.”

In the paper

“When analyzing autophagic flux in vivo, one major limitation is the variability between animals.Different animals do not always activate autophagy at the same time.”

In the paper

“Furthermore, DQ-BSA labeling can be combined with GFP-LC3 to monitor colocalization, and thus visualize the convergence, of amphisomes with a functional degradative compartment (DQ-BSA is internalized by endocytosis).”

In the paper

“In particular, the strong effects of MTOR on protein synthesis may be a confounding factor when analyzing the effects of rapamycin.”

In the paper

“One additional caveat to knockdown experiments is that pathogen-associated molecular pattern (PAMP) recognition pathways can be triggered by double-stranded RNAs (dsRNA), like siRNA probes, or the viral vector systems that deliver shRNA.755”

What the sources say

• ?Not checked — no full text of the sourcecites [755] source

In the paper

“Delivery of these agents by transient transfection, adenovirus, or TAT-mediated protein transduction offers the possibility of their use in cell culture or in vivo.753”

What the sources say

• ?Not checked — no full text of the sourcecites [753] source

In the paper

“Thus, the effect of tamoxifen may differ depending on the cell type.”

In the paper

“When cells are grown in normoxic conditions at high cell density, HIF1A/HIF-1a is stabilized at levels similar to that obtained with low-density cultures under hypoxic conditions.774”

What the sources say

• ?Not checked — no full text of the sourcecites [774] source

In the paper

“For example, trehalose, an MTOR-independent autophagy inducer, 253 can compromise proteasomal activity in cultured primary neurons.739”

What the sources say

• ?Not checked — no full text of the sourcecites [253] source
• ?Not checked — no full text of the sourcecites [739] source