The DOI matches its Crossref record; 48 of the 48 references it cites were found in a registry or an index; none of the 2 sources its claims cite is checked for support yet.
Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy
Open accessAbstract
During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.
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Written from the paper’s full text.
TL;DR
The paper shows that endogenous LC3-II is rapidly degraded by lysosomal hydrolases during starvation-induced autophagy, so the cellular level of LC3-II alone does not reliably indicate autophagic activation. Measuring LC3-II in the presence versus absence of E64d and pepstatin A reveals lysosomal turnover and better estimates autophagy.
Key findings
- Under starvation conditions, endogenous LC3-II was about 3.5-fold higher in HEK293 cells and about 6.7-fold higher in HeLa cells when treated with E64d and pepstatin A than without these inhibitors.
- In HEK293 cells, without inhibitors LC3-II increased 2 h after starvation induction but slightly decreased at 4 h; with E64d and pepstatin A, LC3-II increased dramatically in a time-dependent manner.
- In HeLa cells, LC3-positive puncta and autolysosomes accumulated with E64d and pepstatin A under starvation; autolysosomes per 100 µm² of cytoplasmic area were about 7.3-fold higher with inhibitors than without.
- In A431, MDA-MB-231, and SK-N-SH cells, endogenous LC3-II was present under nutrient-rich conditions despite S6RP phosphorylation, suggesting LC3-II levels are not a simple marker for autophagic activity.
Methods
- HeLa and HEK293 cells were cultured in DMEM with 10% FCS and starved in Krebs-Ringer medium; E64d (10 µg/ml) and pepstatin A (10 µg/ml) were used to inhibit lysosomal proteases.
- Endogenous LC3-I and LC3-II were analyzed by immunoblotting with anti-human LC3 antibody and densitometry; Tor pathway activity was monitored using phospho- and total S6RP, S6K, and Tor antisera.
- LC3 localization was examined by immunofluorescence with Alexa Fluor594-conjugated anti-LC3 antibody and 2D blind deconvolution; autophagosomes/autolysosomes were examined by electron microscopy.
Limitations
- Stated: the anti-human LC3 antibody was not suitable for immunoelectron microscopy, so the relationship between LC3 and autolysosomes could not be directly confirmed by immunoelectron microscopy.
- Stated: there is no evidence for a Cvt-like pathway in mammalian cells, which is offered as one possible explanation for LC3-II under nutrient-rich conditions.
- Evident: the conclusions rely on pharmacological protease inhibitors and cultured cell lines; no in vivo animal validation is described in this paper. The comparison of endogenous LC3 with GFP-human LC3 is cited as unpublished results and is not shown.
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Claims the paper makes
9 Own interpretation · 7 Own result · 2 Sources not found
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In the presence of these inhibitors, LC3-II dramatically accumulates under starvation conditions in both HeLa and HEK293 cells, as do LC3-positive puncta and autolysosomal structures.”
§ Isei > Autophagy 89
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: E64d and pepstatin A inhibit the proteolytic activities of cathepsins B, D and L, causing autolysosomes containing cytosolic components to accumulate.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“When we compared the lysosomal degradation of endogenous LC3 with that of GFP-human LC3, we found that GFP-human LC3 tended to accumulate less than endogenous LC3 in the presence of E64d and pepstatin A under starvation conditions (Tanida I, Ueno T, Kominami E, unpublished results”
§ Isei > Autophagy 89
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In the presence of these inhibitors, LC3-II dramatically accumulates under starvation conditions in both HeLa and HEK293 cells, as do LC3-positive puncta and autolysosomal structures.”
§ Isei > Autophagy 89
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: E64d and pepstatin A inhibit the proteolytic activities of cathepsins B, D and L, causing autolysosomes containing cytosolic components to accumulate.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In the presence of these inhibitors, LC3-II dramatically accumulates under starvation conditions in both HeLa and HEK293 cells, as do LC3-positive puncta and autolysosomal structures.”
§ Isei > Autophagy 89
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: E64d and pepstatin A inhibit the proteolytic activities of cathepsins B, D and L, causing autolysosomes containing cytosolic components to accumulate.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Furthermore, in HeLa cells, LC3-II decreases under starvation conditions in the absence of E64d and pepstatin A, while it increases under the same conditions in HEK293 cells.”
§ Isei > Autophagy 89
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Furthermore, in HeLa cells, LC3-II decreases under starvation conditions in the absence of E64d and pepstatin A, while it increases under the same conditions in HEK293 cells.”
§ Isei > Autophagy 89
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In some cell lines, however, LC3-II remains present under nutrient-rich conditions without inactivation of Tor kinase.”
- Also stated in: § Autophagy 89 · plain
“In some cell lines, including HeLa, A431, MDA-MB-231, and SK-N-SH cells, endogenous LC3-II is present even under nutrientrich conditions without inactivation of Tor kinase.”
§ Isei > Autophagy 89
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Also stated in: § Autophagy 89 · plain
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“These results indicate that the lysosomal degradation of intra-autophagosomal components (Fig. 1, Step 3) significantly affects the amount of endogenous LC3-II after the formation of autophagosomes.”
§ Isei > Autophagy 89
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: E64d and pepstatin A inhibit the proteolytic activities of cathepsins B, D and L, causing autolysosomes containing cytosolic components to accumulate.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Therefore, measurement of endogenous LC3 is better for estimating lysosomal degradation.”
§ Isei > Autophagy 89
plaincorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
In the paper
“Furthermore, inhibiting the fusion of autophagosomes with lysosomes using rab7 RNAi led to the accumulation of LC3-II, indicating that during the impairment of autophagosome-lysosome fusion LC3 is lipidated and LC3-II accumulates.52”
§ Isei > Autophagy 89
What the sources say
- Not matchedcites [52]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Not matched
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Furthermore, these results indicated that the amount of LC3-II does not accurately reflect the activation of starvation-induced autophagy: however, monitoring LC3-II levels in the presence and absence of inhibitors can be used to estimate lysosomal degradation during autophagy.”
§ Isei > Autophagy 89
hedged- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Furthermore, these results indicated that the amount of LC3-II does not accurately reflect the activation of starvation-induced autophagy: however, monitoring LC3-II levels in the presence and absence of inhibitors can be used to estimate lysosomal degradation during autophagy.”
§ Isei > Autophagy 89
plaincorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Therefore, without assaying the lysosomal turnover of LC3-II, the cellular level of LC3-II itself is far from conclusive regarding the activation of autophagy.”
§ Isei > Autophagy 89
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Under the first condition, an increase in endogenous LC3-II may be regarded as a marker for autophagy.”
§ Isei > Autophagy 89
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Thus, to distinguish the first condition from the other two, it is necessary to assay lysosomal turnover of endogenous LC3-II using E64d and pepstatin A.”
§ Isei > Autophagy 89
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Under the latter two conditions, however, an increase in endogenous LC3-II is indicative of the inhibition of autophagic processes (Fig. 1, Steps 2 and 3).”
§ Isei > Autophagy 89
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- It may state two separate claims as one. 2
In the paper
“Furthermore, inhibiting the fusion of autophagosomes with lysosomes using rab7 RNAi led to the accumulation of LC3-II, indicating that during the impairment of autophagosome-lysosome fusion LC3 is lipidated and LC3-II accumulates.52”
§ Isei > Autophagy 89
What the sources say
- Not matchedcites [52]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Not matched
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“However, an increase in the amount of endogenous LC3-II can occur under three conditions: (1) lipidation of LC3-I to form LC3-II during autophagosome-formation, (2) inhibition of the fusion of autophagosomes with lysosomes, and (3) inhibition of lysosomal degradation.”
§ Isei > Autophagy 89
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
The paper relies on this without support.
E64d and pepstatin A inhibit the proteolytic activities of cathepsins B, D and L, causing autolysosomes containing cytosolic components to accumulate.
“When E64d and pepstatin A are added to the medium, the proteolytic activities of these cathepsins are inhibited, and autolysosomes containing cytosolic components accumulate.”
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?
In the citing paper
“LC3 is a cytosolic protein that plays a crucial role in the formation of autophagosomes (Tanida et al., 2005).”
From Nilotinib restores memory function by preventing dopaminergic neuron degeneration in a mouse model of Alzheimer’s Disease (2021) · cites this paper as Tanida et al., 2005
In the citing paper
“Autophagy involves the delivery of cytoplasmic substrates (cellular proteins and damaged organelles) to lysosomes for degradation and impairment of this process is associated with premature ageing and age-associated decline in tissue function [58], [71].”
From Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis (2018) · cites this paper as [58]
In the citing paper
“LC3-II: LC3-I ratio is not a reliable indicator of autophagy in cells possessing dysfunctional lysosomes and so was not evaluated for the senescent cells [58].”
From Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis (2018) · cites this paper as [58]
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.
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Isei > Endogenous LC3-II markedly accumulates in the presence of E64d and pepstatin A during starvation-induced autophagy in HEK293 and HeLa cells.
“Together with our finding that the amount of endogenous LC3-II increased under starvation conditions in the presence of E64d and pepstatin A (Fig. 3B), these results indicate that this increase in LC3-II correlates with an increase in LC3-positive puncta.”
§ Isei > Autophagy 89
“HeLa cells were cultured in DMEM containing 10% FCS (Nutrient-rich, A and B) or transferred to KRB medium for 4 h (Starvation, C, D and G-J).”
Citations
Cites
The publisher hasn’t deposited this paper’s reference list.
Research works printed but not linked: 8
- Ohsumi Y. Molecular dissection of autophagy: Two ubiquitin-like systems. Nat Rev Mol Cell Biol 2001; 2:211-6.
- Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ 2001; 8:569-81.
- Klionsky DJ. Cell biology: Regulated self-cannibalism. Nature 2004; 431:31-2.
- Pfeifer U. Functional morphology of the lysosomal apparatus. In: Glaumann H, Ballard FJ, eds. Lysosomes: Their role in protein breakdown. London: Academic Press, 1987:3-59.
- Seglen PO. Regulation of autophagic protein degradation in isolated liver cells. In: Glaumann H, Ballard FJ, eds. Lysosomes: Their role in protein breakdown. London: Academic Press, 1987:371-414.
- Andersson G, Glaumann H. Current Methods for the isolation of coated vesicles, endo- cytic vesicles, the Golgi complex, lysosomes and autophagic vacuoles. In: Glaumann H, Ballard FJ, eds. Lysosomes: Their role in protein breakdown. London: Academic Press, 1987:61-113.
- Kirschke H, Barrett AJ. Chemistry of lysosomal proteases. In: Glaumann H, Ballard FJ, eds. Lysosomes: Their role in protein breakdown. London: Academic Press, 1987:193-238.
- Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004; 36:2491-502.
Cited by
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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
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InfluentialSelf-citationMethods in Mammalian Autophagy Research
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Guidelines for the use and interpretation of assays for monitoring autophagy
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., et al. (2012) · Autophagy · doi:10.4161/auto.19496 · Cited by 3,996
“The relevant parameter in this assay is the difference in the amount of LC3-II in the presence and absence of saturating levels of inhibitors, which can be used to examine the transit of LC3-II through the autophagic pathway; if flux is occurring, the amount of LC3-II will be higher in the presence of the inhibitor.(1…”
BackgroundInfluentialSelf-citationAutophagy: process and functionPreprint
Mizushima, N. (2007) · Genes & Development · doi:10.1101/gad.1599207 · Cited by 4,104
Regulation mechanisms and signaling pathways of autophagy
He, C., & Klionsky, D. J. (2009) · Annual Review of Genetics · doi:10.1146/annurev-genet-102808-114910 · Cited by 3,609
Metadata
- Authors
- Tanida, I., Minematsu-Ikeguchi, N., Ueno, T., & Kominami, E.
- Affiliations
- Juntendo University (JP)
- Venue
- Autophagy
- Publisher
- Informa UK Limited
- Published
- 2005-07-05
- Volume
- 1
- Issue
- 2
- Pages
- 84-91
- ISSN
- 1554-8627 (ISSN-L)
- 1554-8635
- OpenAlex
- W1990138203
- Semantic Scholar
- 0b28240a0597c546157837f2fb3739b45c71db72
- Cited by
- 1,108
- References
- 0
- Citations by year
- 2026: 27 · 2025: 36 · 2024: 30 · 2023: 42 · 2022: 63 · 2021: 79 · 2020: 67 · 2019: 87 · 2018: 61 · 2017: 70 · 2016: 62 · 2015: 58 · 2014: 54 · 2013: 72 · 2012: 55
- FWCI
- 20.29
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Autophagy (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Japan Private School Promotion Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › 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
- LC3-II, starvation-induced autophagy, autolysosomes, lysosomal proteases, cathepsins, E64d, pepstatin A, autophagosome formation, autophagy markers, LC3 lipidation
- MeSH terms
- Autophagy, Biological Transport, Active, Cell Line, Humans, Hydrolases, Leucine, Lysosomes, Microtubule-Associated Proteins, Pepstatins, Phagosomes, Biomarkers, Microscopy, Electron, Transmission
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