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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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.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
0 of 2 cited sources checked · 0 supported · 0 flagged
- Findingown data
In the presence of E64d and pepstatin A, LC3-positive puncta accumulate under starvation conditions in both HeLa and HEK293 cells.
“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.”
Quote in the paperSubstantial claim- 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.
- Findingown data
GFP-human LC3 tended to accumulate less than endogenous LC3 in the presence of E64d and pepstatin A under starvation conditions.
“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”
Quote in the paperSubstantial claim - Findingown data
In the presence of E64d and pepstatin A, LC3-II dramatically accumulates under starvation conditions in both HeLa and HEK293 cells.
“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.”
Quote in the paperSubstantial claim- 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.
- Findingown data
In the presence of E64d and pepstatin A, autolysosomal structures accumulate under starvation conditions in both HeLa and HEK293 cells.
“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.”
Quote in the paperSubstantial claim- 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.
- Findingown data
In HeLa cells, LC3-II decreases under starvation conditions in the absence of E64d and pepstatin A.
“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.”
Quote in the paperSubstantial claim
- Findingown data
In HEK293 cells, LC3-II increases under starvation conditions in the absence of E64d and pepstatin A.
“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.”
Quote in the paperSubstantial claim - Findingown data
In some cell lines, LC3-II remains present under nutrient-rich conditions without inactivation of Tor kinase.
“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.”
Quote in the paperSubstantial claim - Also stated in: § Autophagy 89 · plain
- Interpretationown data
The lysosomal degradation of intra-autophagosomal components significantly affects the amount of endogenous LC3-II after the formation of autophagosomes.
“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.”
Quote in the paperSubstantial claim- 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.
- Interpretationown data
Measurement of endogenous LC3 is better for estimating lysosomal degradation.
“Therefore, measurement of endogenous LC3 is better for estimating lysosomal degradation.”
Quote in the paperSubstantial claim - Premisecitation
Inhibiting the fusion of autophagosomes with lysosomes using rab7 RNAi led to the accumulation of LC3-II.
“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”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [52]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationown data
The amount of LC3-II does not accurately reflect the activation of starvation-induced autophagy.
“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.”
Quote in the paperSubstantial claim - Interpretationown data
Monitoring LC3-II levels in the presence and absence of inhibitors can be used to estimate lysosomal degradation during autophagy.
“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.”
Quote in the paperSubstantial claim - Interpretationreasoning
Without assaying the lysosomal turnover of LC3-II, the cellular level of LC3-II itself is far from conclusive regarding the activation of autophagy.
“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.”
Quote in the paperSubstantial claim - Interpretationreasoning
Under the first condition (lipidation of LC3-I to form LC3-II during autophagosome formation), an increase in endogenous LC3-II may be regarded as a marker for autophagy.
“Under the first condition, an increase in endogenous LC3-II may be regarded as a marker for autophagy.”
Quote in the paperSubstantial claim - Interpretationreasoning
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.
“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.”
Quote in the paperSubstantial claim - Interpretationreasoning
Under the latter two conditions (inhibition of fusion and inhibition of lysosomal degradation), an increase in endogenous LC3-II is indicative of the inhibition of autophagic processes.
“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).”
Quote in the paperSubstantial claim
- May combine two claims. 2
May combine two claims.
InterpretationcitationDuring the impairment of autophagosome-lysosome fusion, LC3 is lipidated and LC3-II accumulates.
“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”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [52]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
InterpretationreasoningAn 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.
“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.”
Quote in the paperSubstantial claim
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?
LC3 is a cytosolic protein that plays a crucial role in the formation of autophagosomes.
“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
Autophagy involves delivery of cytoplasmic substrates to lysosomes for degradation, and impairment of this process is associated with premature ageing and age-associated decline in tissue function.
“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]
LC3-II:LC3-I ratio is not a reliable indicator of autophagy in cells possessing dysfunctional lysosomes.
“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.
Figure 3B is cited as starvation-condition LC3-II evidence
MinorThe text cites Figure 3B for increased LC3-II under starvation, but the figure caption identifies panel B as a nutrient-rich-condition fluorescence image. The caption assigns starvation conditions to other panels.
“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.”
“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).”
Cites
The publisher hasn’t deposited this paper’s reference list.
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“As another approach, endogenous LC3-I to LC3-II conversion, which is an indicator of autophagosome maturation, was examined by western blotting after rapamycin treatment to induce autophagy [38–40].”
BackgroundLC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells
Jiang, P., & Mizushima, N. (2014) · Methods · doi:10.1016/j.ymeth.2014.11.021 · Cited by 522 · see citing works
Consequences of the selective blockage of chaperone-mediated autophagy
Massey, A. C., Kaushik, S., Sovak, G., Kiffin, R., & Cuervo, A. M. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0507436103 · Cited by 529 · see citing works
The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy
Bartolomeo, S. D., Corazzari, M., Nazio, F., Oliverio, S., Lisi, G., Antonioli, M., Pagliarini, V., Matteoni, S., Fuoco, C., Giunta, L., et al. (2010) · The Journal of Cell Biology · doi:10.1083/jcb.201002100 · Cited by 519 · see citing works
“An important goal for the future will be the identification of the factors responsible of docking the AMBRA1–BECLIN 1 complex on the omegasome compartment, with DFCP1 (or other PI3P-binding proteins) or mCherryLC3 degradation within the lysosome (Tanida et al., 2005).”
BackgroundJagannath, C., Lindsey, D. R., Dhandayuthapani, S., Xu, Y., Hunter, R. L., & Eissa, N. T. (2009) · Nature Medicine · doi:10.1038/nm.1928 · Cited by 486 · see citing works
“1a ), we reasoned that rapamycin increased mycobacterial targeting to the lysosomes, an event that occurs during autophagic flu”
BackgroundAutophagosome Formation and Molecular Mechanism of Autophagy
Tanida, I. (2010) · Antioxidants and Redox Signaling · doi:10.1089/ars.2010.3482 · Cited by 489 · see citing works
“Therefore, LC3-II itself is used as a marker of autophagosome formation and its degradation to monitor late autophagic flux (102).”
Fader, C. M., Sánchez‐Martínez, D., Furlán, M., & Colombo, M. I. (2007) · Traffic · doi:10.1111/j.1600-0854.2007.00677.x · Cited by 470 · see citing works
“As indicated above, conditions that stimulate autophagy lead to an increased processing of LC3, which can be assessed by Western blot (28).”
BackgroundLee, S., Satō, Y., & Nixon, R. A. (2011) · Journal of Neuroscience · doi:10.1523/jneurosci.6412-10.2011 · Cited by 471 · see citing works
“DOI:10.1523/JNEUROSCI.6412-10.2011 Copyright © 2011 the authors 0270-6474/11/317817-14$15.00/0 somes to form autolysosomes (Tanida et al., 2005).”
BackgroundInfluentialNecrotic, apoptotic and autophagic cell fates triggered by nanoparticles
Mohammadinejad, R., Moosavi, M. A., Tavakol, S., Vardar, D. Ö., Hosseini, A., Rahmati, M., Dini, L., Hussain, S., Mandegary, A., & Klionsky, D. J. (2018) · Autophagy · doi:10.1080/15548627.2018.1509171 · Cited by 464 · see citing works
“The cargo is broken down by hydrolases and the products are released into the cytosol through permeases [66].”
BackgroundAutophagy and multivesicular bodies: two closely related partners
Fader, C. M., & Colombo, M. I. (2008) · Cell Death and Differentiation · doi:10.1038/cdd.2008.168 · Cited by 451 · see citing works
Masaldan, S., Clatworthy, S. A. S., Gamell, C., Meggyesy, P. M., Rigopoulos, A.-T., Haupt, S., Haupt, Y., Denoyer, D., Adlard, P. A., Bush, A. I., et al. (2018) · Redox Biology · doi:10.1016/j.redox.2017.08.015 · Cited by 452 · see citing works
The Itinerary of Autophagosomes: From Peripheral Formation to Kiss‐and‐Run Fusion with Lysosomes
Jahreiss, L., Menzies, F. M., & Rubinsztein, D. C. (2008) · Traffic · doi:10.1111/j.1600-0854.2008.00701.x · Cited by 453 · see citing works
“It has previously been reported that GFP–LC3 fluorescence is lost rapidly upon autophagosome–lysosome fusion (15), because of both degradation and the low pH in the lysosome, as GFP itself has a pKa of about 6.”
BackgroundZhu, J.-H., Horbinski, C., Guo, F.-L., Watkins, S. C., Uchiyama, Y., & Chu, C. T. (2007) · American Journal of Pathology · doi:10.2353/ajpath.2007.060524 · Cited by 469 · see citing works
“A method to assess AV induction is to measure LC3-II levels in the presence of an inhibitor of AV fusion/ degradation.(51) MPP caused increased LC3-II compared with control cells in the presence of bafilomycin A1, an inhibitor of early AV acidification and fusion to lysosomes at doses 10 nmol/L(52,53) (Figure 3A, comp…”
MethodFunctional interaction between autophagy and ciliogenesis
Pampliega, O., Orhon, I., Patel, B., Sridhar, S., Díaz-Carretero, A., Beau, I., Codogno, P., Satir, B. H., Satir, P., & Cuervo, A. M. (2013) · Nature · doi:10.1038/nature12639 · Cited by 424 · see citing works
Alegre‐Abarrategui, J., Christian, H., Lufino, M. M., Dafinca, R., Venda, L. L., Ansorge, O., & Wade‐Martins, R. (2009) · Human Molecular Genetics · doi:10.1093/hmg/ddp346 · Cited by 433 · see citing works
“Since LC3-II is itself degraded by autophagy we determined autophagic activity by comparing LC3-II levels in the presence or absence of 200 nM bafilomycin A1 (BFA), a specific inhibitor of autophagic degradation (27) under starvation conditions [LC3-II turnover (28)].”
MethodAcetylation Targets Mutant Huntingtin to Autophagosomes for Degradation
Jeong, H., Then, F., Melia, T. J., Mazzulli, J. R., Cui, L., Savas, J. N., Voisine, C., Paganetti, P., Tanese, N., Hart, A. C., et al. (2009) · Cell · doi:10.1016/j.cell.2009.03.018 · Cited by 419 · see citing works
“Next, we examined clearance of acetylated mutant Htt by monitoring autophagic activity in live cells by LC3, a mammalian homolog of yeast Atg8 that gets incorporated into autophagosomal (AV) membrane (Kabeya et al., 2000; Tanida et al., 2005).”
Scarlatti, F., Maffei, R., Beau, I., Codogno, P., & Ghidoni, R. (2008) · Cell Death & Differentiation · doi:10.1038/cdd.2008.51 · Cited by 412 · see citing works
The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy
Webster, C. P., Smith, E. F., Bauer, C. S., Moller, A., Hautbergue, G. M., Ferraiuolo, L., Myszczynska, M. A., Higginbottom, A., Walsh, M. J., Whitworth, A. J., et al. (2016) · The EMBO Journal · doi:10.15252/embj.201694401 · Cited by 404 · see citing works
“Under these conditions, counting autophagosomes allows specific quantification of the induction of autophagy (Tanida et al , 2005).”
Majumder, S., Richardson, A. G., Strong, R., & Oddo, S. (2011) · PLoS ONE · doi:10.1371/journal.pone.0025416 · Cited by 401 · see citing works
“[65].”
Jiang, M., Liu, K., Luo, J., & Dong, Z. (2010) · American Journal Of Pathology · doi:10.2353/ajpath.2010.090594 · Cited by 375 · see citing works
Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy.
Adhami, F., Liao, G., Morozov, Y., Schloemer, A., Schmithorst, V., Lorenz, J., Dunn, R., Vorhees, C., Wills-Karp, M., Degen, J., et al. (2006) · American Journal of Pathology · doi:10.2353/ajpath.2006.051066 · Cited by 386 · see citing works
Application and interpretation of current autophagy inhibitors and activators
Yang, Y.-P., Hu, L.-F., Zheng, H.-F., Mao, C., Hu, W.-D., Xiong, K., Wang, F., & Liu, C.-F. (2013) · Acta Pharmacologica Sinica · doi:10.1038/aps.2013.5 · Cited by 373 · see citing works
An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure
Takahashi, Y., He, H., Tang, Z., Hattori, T., Liu, Y., Young, M. M., Serfass, J. M., Chen, L., Gebru, M. T., Chen, C., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-05254-w · Cited by 365 · see citing works
Dysfunctional autophagy in RPE, a contributing factor in age-related macular degeneration
Golestaneh, N., Chu, Y., Xiao, Y.-Y., Stoleru, G. L., & Theos, A. C. (2017) · Cell Death & Disease · doi:10.1038/cddis.2016.453 · Cited by 357 · see citing works
“Lysosomal inhibitors E64d and Pepstatin A were added in the last 2 h of starvation at a concentration of 10 μg/ml and the cells were lysed and analyzed with immunoblot for LC3 and p62 based on the established protocol.(35)”
MethodKatayama, M., Kawaguchi, T., Berger, M. S., & Pieper, R. O. (2006) · Cell Death and Differentiation · doi:10.1038/sj.cdd.4402030 · Cited by 353 · see citing works
“To assess the autophagic processing of MAP1-LC3, the cells were collected separately from the samples above by a modified method as described previously.(24) Briefly, 3 h prior to collection of the samples, E64d (10 mg/ml) and pepstatin A (10mg/ml) were added.”
Sato, K., Tsuchihara, K., Fujii, S., Sugiyama, M., Goya, T., Atomi, Y., Ueno, T., Ochiai, A., & Esumi, H. (2007) · Cancer Research · doi:10.1158/0008-5472.can-07-1462 · Cited by 352 · see citing works
“The membrane-bound LC3-II protein level is regarded as a marker for the formation of autophagosomes (9, 23).”
BackgroundCarra, S., Seguin, S. J., Lambert, H., & Landry, J. (2007) · Journal of Biological Chemistry · doi:10.1074/jbc.m706304200 · Cited by 347 · see citing works
Piao, M. J., Ahn, M. J., Kang, K. A., Ryu, Y. S., Hyun, Y. J., Shilnikova, K., Zhen, A. X., Jeong, J. W., Choi, Y. H., Kang, H. K., et al. (2018) · Archives of Toxicology · doi:10.1007/s00204-018-2197-9 · Cited by 339 · see citing works
“The two distinct steps of autophagy, autophagosome formation and autolysosome formation, can be discerned by the presence of LC3-phospholipid conjugates (Tanida et al. 2005).”
Fujita, E., Kouroku, Y., Isoai, A., Kumagai, H., Misutani, A., Matsuda, C., Hayashi, Y. K., & Momoi, T. (2007) · Human Molecular Genetics · doi:10.1093/hmg/ddm002 · Cited by 342 · see citing works
“Inhibition of lysosomal proteases, E64d/pepstatin A ( 42 ), and Atg5 deficiency stimulated mutant (L1341P)-dysferlin aggregation in the mammalian cells (Figs 4 and 6), suggesting that the autophagy/lysosome pathway, an alternative ERAD system, is further stimulated by mutant dysferlin aggregates in the ER for their de…”
BackgroundInfluentialPasquier, B. (2016) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-015-2104-y · Cited by 329 · see citing works
Koike, M., Shibata, M., Waguri, S., Yoshimura, K., Tanida, I., Kominami, E., Gotow, T., Peters, C., von Figura, K., Mizushima, N., et al. (2005) · The American Journal of Pathology · doi:10.1016/s0002-9440(10)61253-9 · Cited by 330 · see citing works
“Moreover, the increase in the LC3 level by such proteinase inhibitors does not occur under nutrient-rich conditions.(55,56) From these lines of evidence, it is possible to assume that the significant increases in amounts of LC3-II in cathepsin-deficient brains at each stage are attributed to the fact that autophagy is…”
BackgroundSelf-citationProteolytic and lipolytic responses to starvation
Finn, P. F., & Dice, J. F. (2006) · Nutrition · doi:10.1016/j.nut.2006.04.008 · Cited by 321 · see citing works
Khandia, R., Dadar, M., Munjal, A. K., Dhama, K., Karthik, K., Tiwari, R., Yatoo, M. I., Iqbal, H. M. N., Singh, K. P., Joshi, S. K., et al. (2019) · Cells · doi:10.3390/cells8070674 · Cited by 323 · see citing works
“Cells treated with E64d and pepstatin A exhibit increased LC3-II levels [475].”
BackgroundInduction of Autophagy in Axonal Dystrophy and Degeneration
Wang, Q. J., Ding, Y., Kohtz, S., Mizushima, N., Cristea, I. M., Rout, M. P., Chait, B. T., Zhong, Y., Heintz, N., & Yue, Z. (2006) · The Journal of Neuroscience · doi:10.1523/jneurosci.2261-06.2006 · Cited by 308 · see citing works
“To examine autophagy induction, we monitored the level of phosphorylation of the S6 ribosomal protein (Ravikumar et al., 2004; Tanida et al., 2005).”
BackgroundMethodWhy should autophagic flux be assessed?
Zhang, X.-J., Chen, S., Huang, K.-X., & Le, W.-D. (2013) · Acta Pharmacologica Sinica · doi:10.1038/aps.2012.184 · Cited by 304 · see citing works
FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells
Bell, B. D., Leverrier, S., Weist, B. M., Newton, R. H., Arechiga, A. F., Luhrs, K. A., Morrissette, N. S., & Walsh, C. M. (2008) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0808597105 · Cited by 290 · see citing works
“Treatment with the lysosomal protease inhibitors E64d and pepstatin A (pepA) for the last four hours of culture in WT and FADDdd T cells enhanced the overall detection of LC3, showing that this increase was not a consequence of defective fusion and subsequent degradation of autophagosomes within lysosomes (32) (Fig.”
BackgroundKiyono, K., Suzuki, H. I., Matsuyama, H., Morishita, Y., Komuro, A., Kano, M. R., Sugimoto, K., & Miyazono, K. (2009) · Cancer Research · doi:10.1158/0008-5472.can-08-4401 · Cited by 285 · see citing works
“We next determined the autophagy flux by LC3 turnover assay, which measures the amount of LC3-II delivered to the lysosomes by comparing the LC3-II amounts in the presence and absence of lysosomal inhibitors (29, 30).”
Liver autophagy contributes to the maintenance of blood glucose and amino acid levels
Ezaki, J., Matsumoto, N., Takeda‐Ezaki, M., Komatsu, M., Takahashi, K., Hiraoka, Y., Taka, H., Fujimura, T., Takehana, K., Yoshida, M., et al. (2011) · Autophagy · doi:10.4161/auto.7.7.15371 · Cited by 282 · see citing works
The Upstream Pathway of mTOR-Mediated Autophagy in Liver Diseases
Wang, H., Liu, Y., Wang, D., Xu, Y., Dong, R., Yang, Y., Lv, Q., Chen, X., & Zhang, Z. (2019) · Cells · doi:10.3390/cells8121597 · Cited by 281 · see citing works
Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome
Fujii, S., Mitsunaga, S., Yamazaki, M., Hasebe, T., Ishii, G., Kojima, M., Kinoshitá, T., Ueno, T., Esumi, H., & Ochiai, A. (2008) · Cancer Science · doi:10.1111/j.1349-7006.2008.00893.x · Cited by 279 · see citing works
Regulation of Autophagy by the p300 Acetyltransferase
Lee, I. H., & Finkel, T. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m807135200 · Cited by 276 · see citing works
Macroautophagy Regulates Energy Metabolism during Effector T Cell Activation
Hubbard, V. M., Valdor, R., Patel, B., Singh, R., Cuervo, A. M., & Macian, F. (2010) · The Journal of Immunology · doi:10.4049/jimmunol.1000576 · Cited by 275 · see citing works
The regulation of autophagy by calcium signals: Do we have a consensus?
Bootman, M. D., Chehab, T., Bultynck, G., Parys, J. B., & Rietdorf, K. (2017) · Cell Calcium · doi:10.1016/j.ceca.2017.08.005 · Cited by 271 · see citing works
Photoreceptor cell death and rescue in retinal detachment and degenerations
Murakami, Y., Notomi, S., Hisatomi, T., Nakazawa, T., Ishibashi, T., Miller, J. W., & Vavvas, D. G. (2013) · Progress in Retinal and Eye Research · doi:10.1016/j.preteyeres.2013.08.001 · Cited by 248 · see citing works
“Similarly, the increased levels of LC3-II expression represent either the increases in LC3 conversion and/or the decreased degradation of LC3-II in the autolysosomes (Tanida et al., 2005).”
Shin, J. Y., Park, H. J., Kim, H. N., Oh, S. H., Bae, J.-S., Ha, H.-J., & Lee, P. H. (2014) · Autophagy · doi:10.4161/auto.26508 · Cited by 243 · see citing works
“Therefore, the amount of LC3-II is indicative of the number of autophagosomes and serves as a good marker of autophagosome formation.(24) SH-SY5Y cells were treated with different concentrations to evaluate the direct effects of Aβ (Aβ 1–42 ).”
BackgroundFortunato, F., Bürgers, H. F., Bergmann, F., Rieger, P., Büchler, M. W., Kroemer, G., & Werner, J. (2009) · Gastroenterology · doi:10.1053/j.gastro.2009.04.003 · Cited by 246 · see citing works
Vázquez‐Martín, A., Oliveras‐Ferraros, C., & Menéndez, J. A. (2009) · PLoS ONE · doi:10.1371/journal.pone.0006251 · Cited by 245 · see citing works
“the product of the autophagic posttranslational modification of LC3- correlates with the number of autophagosomes [45–49].”
BackgroundZhou, X., Wang, L., Hasegawa, H., Amin, P., Han, B.-X., Kaneko, S., He, Y.-W., & Wang, F. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0914725107 · Cited by 236 · see citing works
Oxidative Stress and Mitochondrial Dysfunction in Chronic Kidney Disease
Ho, H.-J., & Shirakawa, H. (2022) · Cells · doi:10.3390/cells12010088 · Cited by 255 · see citing works
“In this complex process, the LC3-II/I ratio and p62 protein are central autophagy-related factors that are ubiquitously used as autophagy markers to measure autophagic activity [82].”
BackgroundAutophagy and oxidative stress in cardiovascular diseases
Mei, Y., Thompson, M. D., Cohen, R. A., & Tong, X. (2014) · Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease · doi:10.1016/j.bbadis.2014.05.005 · Cited by 243 · see citing works
Xie, C., Chan, W. Y., Yu, S. S. B., Zhao, J., & Cheng, C. H. K. (2011) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2011.06.016 · Cited by 242 · see citing works
“We also studied the autophagic flux after bufalin treatment, which is a more accurate reflection of the autophagic activity [29,30].”
BackgroundKandalepas, P. C., Sadleir, K. R., Eimer, W. A., Zhao, J., Nicholson, D. A., & Vassar, R. (2013) · Acta Neuropathologica · doi:10.1007/s00401-013-1152-3 · Cited by 240 · see citing works
Trehalose protects against oxidative stress by regulating the Keap1–Nrf2 and autophagy pathways
Mizunoe, Y., Kobayashi, M., Sudo, Y., Watanabe, S., Yasukawa, H., Natori, D., Hoshino, A., Negishi, A., Okita, N., Komatsu, M., et al. (2017) · Redox Biology · doi:10.1016/j.redox.2017.09.007 · Cited by 242 · see citing works
“As p62 protein accumulation was observed in parallel with suppression of autophagy, p62 is also commonly used as a marker of autophagic degradation [7,8].”
BackgroundRoot, J. T., Merino, P., Nuckols, A., Johnson, M. A., & Kukar, T. L. (2021) · Neurobiology of Disease · doi:10.1016/j.nbd.2021.105360 · Cited by 230 · see citing works
The role of mTOR signaling in Alzheimer disease
Oddo, S. (2012) · Frontiers in Bioscience-Scholar · doi:10.2741/s310 · Cited by 235 · see citing works
“After its activation, LC3-I is lipidated to form membraneassociated LC3-II, which is incorporated in the growing autophagosome membrane and is often used as a marker of autophagy induction (94, 95).”
BackgroundTanida, I. (2010) · Microbiology and Immunology · doi:10.1111/j.1348-0421.2010.00271.x · Cited by 238 · see citing works
“A mixture of E64d (a membrane-permeable inhibitor of cathepsins B, H, and L) and pepstatin A (a membrane-permeable inhibitor of cathepsins D and E) is used to inhibit lysosomal function (91).”
MethodExcess Peroxisomes Are Degraded by Autophagic Machinery in Mammals
Iwata, J.-I., Ezaki, J., Komatsu, M., Yokota, S., Ueno, T., Tanida, I., Chiba, T., Tanaka, K., & Kominami, E. (2005) · Journal of Biological Chemistry · doi:10.1074/jbc.m512283200 · Cited by 236 · see citing works
Chapter 1 Monitoring Autophagy in Mammalian Cultured Cells through the Dynamics of LC3
Kimura, S., Fujita, N., Noda, T., & Yoshimori, T. (2009) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(08)03601-x · Cited by 236 · see citing works
The Spectrum of Achalasia: Lessons From Studies of Pathophysiology and High-Resolution Manometry
Kahrilas, P. J., & Boeckxstaens, G. E. (2013) · Gastroenterology · doi:10.1053/j.gastro.2013.08.038 · Cited by 234 · see citing works
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“considered to be the fusion of autophagosomes and lysosomes [53].”
BackgroundActive Ras Triggers Death in Glioblastoma Cells through Hyperstimulation of Macropinocytosis
Overmeyer, J. H., Kaul, A., Johnson, E. E., & Maltese, W. A. (2008) · Molecular Cancer Research · doi:10.1158/1541-7786.mcr-07-2036 · Cited by 229 · see citing works
“the levels of LC3-II in the presence and absence of lysosomal protease inhibitors (20).”
BackgroundChemical modulation of chaperone-mediated autophagy by retinoic acid derivatives
Anguiano, J., Garner, T. P., Mahalingam, M., Das, B. C., Gavathiotis, E., & Cuervo, A. M. (2013) · Nature Chemical Biology · doi:10.1038/nchembio.1230 · Cited by 226 · see citing works
The Pancreatitis-induced Vacuole Membrane Protein 1 Triggers Autophagy in Mammalian Cells
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Liao, G., Yao, Y., Liu, J., Yu, Z., Cheung, S., Xie, A., Liang, X., & Bi, X. (2007) · The American Journal of Pathology · doi:10.2353/ajpath.2007.070052 · Cited by 220 · see citing works
- 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
- 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
- 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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