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How to Interpret LC3 Immunoblotting
Open accessAbstract
Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.
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“LC3, which is a post-translational modifier, is required for autophagosome formation (Mizushima and Yoshimori, 2007).”
From Inducible Rpt3, a Proteasome Component, Knockout in Adult Skeletal Muscle Results in Muscle Atrophy (2020) · cites this paper as Mizushima and Yoshimori, 2007
In the citing paper
“In neurons ATG9 localizes to axons and presynaptic sites, and requires active transport by the kinesin motor KIF1A to direct its localization into distal neurites [30,537].”
From Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1 (2021) · cites this paper as [30]
Citations
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InfluentialSelf-citationMethods in Mammalian Autophagy Research
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BackgroundMethodInfluentialSelf-citationAutophagy: process and functionPreprint
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“One current problem is that this degradation step is rather difficult to measure, although some methods to monitor autophagy flux have been proposed (Tanida et al. 2005; Klionsky et al. 2007; Mizushima and Yoshimori 2007).”
BackgroundSelf-citationAntimicrobial Autophagy: A Conserved Innate Immune Response in Drosophila
Moy, R. H., & Cherry, S. R. (2013) · Journal of Innate Immunity · doi:10.1159/000350326 · Cited by 3,551
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Self-citationGuidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
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Nutrient-dependent mTORC1 Association with the ULK1–Atg13–FIP200 Complex Required for Autophagy
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“Because LC3-II inside autophagosomes is degraded in autolysosomes, we could see accumulation of LC3-II more clearly if cells were treated with lysosome protease inhibitors (E64d and pepstatin A; Figure Vol. 20, April 1, 2009 1983 3A, lane 3; Tanida et al., 2005; Mizushima and Yoshimori, 2007).”
BackgroundMethodSelf-citationULK-Atg13-FIP200 Complexes Mediate mTOR Signaling to the Autophagy Machinery
Jung, C. H., Jun, C. B., Ro, S.-H., Kim, Y.-M., Otto, N. M., Cao, J., Kundu, M., & Kim, D.-H. (2009) · Molecular Biology of the Cell · doi:10.1091/mbc.e08-12-1249 · Cited by 2,025
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BackgroundMethodInfluentialAutophagosomes form at ER–mitochondria contact sites
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Self-citationNetwork organization of the human autophagy system
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“Third, we used bafilomycin A1, a specific inhibitor of vacuolar H -ATPases and a blocker of autophagosome-lysosome fusion (20, 31).”
MethodBeclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG
Itakura, E., Kishi, C., Inoue, K., & Mizushima, N. (2008) · Molecular Biology of the Cell · doi:10.1091/mbc.e08-01-0080 · Cited by 1,176
“We also monitored the expression level of autophagy substrate p62, another indicator of autophagic degradation (Bjørkøy et al., 2005; Mizushima and Yoshimori, 2007).”
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Monitoring and Measuring Autophagy
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“Also, LC3-II can ectopically localize on non-autophagosome structures that are not turned over in the lysosome [8,15,32].”
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“While LC3-I is present in the cytosol, the lipidated LC3-II form is associated mainly with autophagosomal membranes, serving as a good indicator of autophagosomes (32, 33).”
BackgroundInfluentialCárdenas, C., Miller, R. A., Smith, I., Bui, T., Molgó, J., Müller, M., Vais, H., Cheung, K.-H., Yang, J., Parker, I., et al. (2010) · Cell · doi:10.1016/j.cell.2010.06.007 · Cited by 1,026
“Amount of LC3-II was normalized as LC3-I/(LC3-I+LC3-II), described as the most accurate quantitative method (Mizushima and Yoshimori, 2007).”
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells
Hara, T., Takamura, A., Kishi, C., Iemura, S.-I., Natsume, T., Guan, J.-L., & Mizushima, N. (2008) · The Journal of Cell Biology · doi:10.1083/jcb.200712064 · Cited by 904
“…numerous autophagic vacuoles, which occupied 3% of the total cytoplasmic volume ( 2% autophagosomes To monitor the autophagic ability of FIP200 / MEFs more precisely, we determined the autophagy fl ux in these cells by the LC3 turnover assay ( Tanida et al., 2005 ; Mizushima and Yoshimori, 2007 ).”
BackgroundMethodSelf-citationEndogenous HMGB1 regulates autophagy
Tang, D., Kang, R., Livesey, K. M., Cheh, C.-W., Farkas, A., Loughran, P., Hoppe, G., Bianchi, M. E., Tracey, K. J., Zeh, H. J., et al. (2010) · Journal of Cell Biology · doi:10.1083/jcb.200911078 · Cited by 951
“(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3.”
BackgroundInfluentialPathogenic Lysosomal Depletion in Parkinson's Disease
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“Because LC3II is normally degraded by lysosomal proteases once the AP fuses with a lysosome to form an autophagolysosome (Mizushima and Yoshimori, 2007), the accumulation of LC3II protein observed following MPTP intoxication indicates that, in addition to any potential induction of new AP…”
BackgroundAutophagy-Dependent Ferroptosis: Machinery and Regulation
Liu, J., Kuang, F., Kroemer, G., Klionsky, D. J., Kang, R., & Tang, D. (2020) · Cell chemical biology · doi:10.1016/j.chembiol.2020.02.005 · Cited by 819
“At the same time, MAP1LC3-II can be degraded in a lysosome-dependent manner (Mizushima and Yoshimori, 2007; Pankiv et al., 2007).”
BackgroundSuperoxide is the major reactive oxygen species regulating autophagy
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Autophagy Controls IL-1β Secretion by Targeting Pro-IL-1β for Degradation
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Autophagy: assays and artifacts
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Vitamin D3 Induces Autophagy in Human Monocytes/Macrophages via Cathelicidin
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Autophagy regulates adipose mass and differentiation in mice
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Toll‐like receptors control autophagy
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“Immunoblot analysis of LC3 bands was carried out in the presence of Bafilomycin A1 (Mizushima and Yoshimori, 2007), an inhibitor of autophagic degradation/flux.”
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Apoptosis and necrosis: Detection, discrimination and phagocytosis
Krysko, D. V., Berghe, T. V., D'Herde, K. G. M. A., & Vandenabeele, P. (2008) · Methods · doi:10.1016/j.ymeth.2007.12.001 · Cited by 707
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“In addition, we observed decreased levels of lipidated LC3-II by Western blot upon engulfment of dead cells by ATG7-deficient macrophages (Fig.”
BackgroundInfluentialRetractedCharacterization of ferroptosis in murine models of hemochromatosis
Wang, H., An, P., Xie, E., Wu, Q., Fang, X., Gao, H., Zhang, Z., Li, Y., Wang, X., Zhang, J., et al. (2017) · Hepatology · doi:10.1002/hep.29117 · Cited by 662
“However, LC3-II and p62, two markers used to measure autophagy,((40)) were not increased in our HH mice or in HID-fed Slc7a11 mice.”
Ma, X., Wu, Y., Jin, S., Tian, Y., Zhang, X., Zhao, Y., Yu, L., & Liang, X. (2011) · ACS Nano · doi:10.1021/nn202155y · Cited by 642
“org 8632 inhibitor, we can determine whether or not autophagy is, in fact, induced.(32) Starvation is the stimulus most widely used to induce autophagy, and we found that”
BackgroundMorselli, E., Maiuri, M. C., Markaki, M., Megalou, E., Pasparaki, A., Palikaras, K., Criollo, A., Galluzzi, L., Malik, S. A., Vitale, I., et al. (2010) · Cell Death & Disease · doi:10.1038/cddis.2009.8 · Cited by 613
The dynamic nature of autophagy in cancerPreprint
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“The lipidated (LC3-II) form can be detected as a faster-migrating band by immunoblotting (Mizushima and Yoshimori 2007).”
BackgroundRoca, H., Varsos, Z. S., Sud, S., Craig, M. J., Chi, Y., & Pienta, K. J. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.042671 · Cited by 587
Autophagy and oxidative stress associated with gold nanoparticles
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“05) which correlates with autophagosome formation [17].”
LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells
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An Atg4B Mutant Hampers the Lipidation of LC3 Paralogues and Causes Defects in Autophagosome Closure
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“As it has been reported that p62/SQSTM1 is a selective substrate of autophagy (Bjorkoy et al., 2005; Mizushima and Yoshimori, 2007), we investigated autophagic degradation by monitoring endogenous p62 protein levels.”
BackgroundSelf-citationProtein quality control during aging involves recruitment of the macroautophagy pathway by BAG3
Gamerdinger, M., Hajieva, P., Kaya, A. M., Wolfrum, U., Hartl, F. U., & Behl, C. (2009) · The EMBO Journal · doi:10.1038/emboj.2009.29 · Cited by 522
“A well-established marker for autophagy is LC3-II, and the autophagic flux can be measured by monitoring LC3-II accumulation in the presence of lysosomal inhibitors (Mizushima and Yoshimori, 2007).”
BackgroundAutophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages
Kyei, G. B., Dinkins, C., Davis, A. S., Roberts, E., Singh, S. B., Dong, C., Wu, L., Kominami, E., Ueno, T., Yamamoto, A., et al. (2009) · Journal of Cell Biology · doi:10.1083/jcb.200903070 · Cited by 509
“We first examined whether Nef affected the maturation (degradative) to the lipidated LC3-II form, which shows increased electrophoretic mobility (Kabeya et al., 2000; Mizushima and Yoshimori, 2007).”
BackgroundMethodInfluentialReduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson’s disease
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“LC3-I to LC3-II conversion, a third autophagy-associated change (19), was seen in hypoxic T98G (Fig.”
BackgroundApoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL
Luo, S., & Rubinsztein, D. C. (2009) · Cell Death and Differentiation · doi:10.1038/cdd.2009.121 · Cited by 495
“Endogenous LC3-I levels appear very low in HeLa cells as LC3 antibody has stronger immunoreactivity to LC3-II than LC3 I.(33) (c) GFP-LC3 was co-transfected with increasing levels of Bax plasmid, as indicated in duplicate.”
Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner
Frank, M., Duvezin-Caubet, S., Koob, S., Occhipinti, A., Jagasia, R., Petcherski, A., Ruonala, M. O., Priault, M., Salin, B., & Reichert, A. S. (2012) · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research · doi:10.1016/j.bbamcr.2012.08.007 · Cited by 468
“As a control we induced bulk autophagy by starvation, a condition known to increase autophagic flux [31,56,61].”
BackgroundThe Roles of Therapy-Induced Autophagy and Necrosis in Cancer Treatment
Amaravadi, R. K., & Thompson, C. B. (2007) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-07-1595 · Cited by 474
HMGB1 release and redox regulates autophagy and apoptosis in cancer cells
Tang, D., Kang, R., Cheh, C.-W., Livesey, K. M., Liang, X., Schapiro, N. E., Benschop, R., Sparvero, L. J., Amoscato, A. A., Tracey, K. J., et al. (2010) · Oncogene · doi:10.1038/onc.2010.261 · Cited by 461
“Microtubule-associated protein light chain 3 (LC3) is now widely used as a marker to monitor autophagy (Mizushima and Yoshimori, 2007).”
BackgroundMethodMasaldan, 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
“LC3-II localizes to autophagosomes that ultimately fuse with lysosomes (autolysosomes) and is consequently degraded by lysosomal hydrolases [58,59].”
BackgroundAMPK–SKP2–CARM1 signalling cascade in transcriptional regulation of autophagy
Shin, H.-J. R., Kim, H., Oh, S., Lee, J.-G., Kee, M., Ko, H.-J., Kweon, M.-N., Won, K.-J., & Baek, S. H. (2016) · Nature · doi:10.1038/nature18014 · Cited by 417
Molecular machinery and interplay of apoptosis and autophagy in coronary heart disease
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ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy
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Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence
Ramírez, L. V., Vodnala, S. K., Nini, R., Hunter, K. W., & Green, J. E. (2018) · Nature Communications · doi:10.1038/s41467-018-04070-6 · Cited by 433
PGC-1α-mediated regulation of mitochondrial function and physiological implications
Halling, J. F., & Pilegaard, H. (2020) · Applied Physiology, Nutrition, and Metabolism · doi:10.1139/apnm-2020-0005 · Cited by 430
“In addition, lipidation of light chain (LC)3I to LC3II is required for autophagosome maturation (Mizushima & Komatsu, 2011), which is why the content of LC3II and the ratio of LC3II/LC3I are often used as markers of autophagy activation (Kabeya et al., 2000;Mizushima & Yoshimori, 2007).”
BackgroundAutophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome
Cho, M.-H., Cho, K., Kang, H.-J., Jeon, E.-Y., Kim, H.-S., Kwon, H.-J., Kim, H.-M., Kim, D.-H., & Yoon, S.-Y. (2014) · Autophagy · doi:10.4161/auto.29647 · Cited by 395
“bBraak stage based on neurofibrillary tangles was 5 or 6 in aD cases, and 0 or 1 in controls.(25) cBraak stage based on amyloid plaques was c in aD cases, and 0 or a in controls.”
BackgroundChloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy
Maycotte, P., Aryal, S., Cummings, C. T., Thorburn, J., Morgan, M. J., & Thorburn, A. M. (2012) · Autophagy · doi:10.4161/auto.8.2.18554 · Cited by 406
“However, LC3II is also degraded inside the lysosomes or de-conjugated by Atg4.(4,24) Therefore, in order to measure autophagic flux, lysosomal inhibitors (CQ, Baf A, pepstatin A/E64D) are used in order to inhibit LC3II degradation, since in the absence of these inhibitors, an autophagy-inducing treatment can result in…”
BackgroundA role for autophagy during hepatic stellate cell activation
Thoen, L. F. R., Guimarães, E. L. M., Dollé, L., Mannaerts, I., Najimi, M., Sokal, E., & van Grunsven, L. A. (2011) · Journal of Hepatology · doi:10.1016/j.jhep.2011.07.010 · Cited by 385
“We first investigated the expression of LC3B, a protein involved in autophagosome formation [14], in total liver samples of healthy and 4 week CCl4-treated mice (Fig.”
BackgroundTanaka, S., Hikita, H., Tatsumi, T., Sakamori, R., Nozaki, Y., Sakane, S., Shiode, Y., Nakabori, T., Saito, Y., Hiramatsu, N., et al. (2016) · Hepatology · doi:10.1002/hep.28820 · Cited by 376
“To confirm this hypothesis, we compared LC3-II expression levels in the presence and absence of bafilomycin, which inhibits lysosomal enzyme activity as well as the fusion of autophagosomes with lysosomes.((23)) Bafilomycin clearly increased levels of LC3-II expression in control HepG2 cells.”
BackgroundMethodSelf-citationLoss of the Parkinson's disease-linked gene DJ-1 perturbs mitochondrial dynamics
Irrcher, I., Aleyasin, H., Seifert, E. L., Hewitt, S. J., Chhabra, S., Phillips, M., Lutz, A. K., Rousseaux, M. W. C., Bevilacqua, L., Jahani‐Asl, A., et al. (2010) · Human Molecular Genetics · doi:10.1093/hmg/ddq288 · Cited by 394
Jiang, M., Liu, K., Luo, J., & Dong, Z. (2010) · American Journal Of Pathology · doi:10.2353/ajpath.2010.090594 · Cited by 380
Ebrahimi‐Fakhari, D., Cantuti‐Castelvetri, I., Fan, Z., Rockenstein, E. M., Masliah, E., Hyman, B. T., McLean, P. J., & Unni, V. K. (2011) · Journal of Neuroscience · doi:10.1523/jneurosci.1560-11.2011 · Cited by 387
“7A,B), indicating an accumulation of autophagosomes consistent with a block in autophagic clearance by BafA1 (Mizushima and Yoshimori, 2007; Klionsky et al., 2008; Mizushima et al., 2010).”
BackgroundKrebiehl, G., Ruckerbauer, S., Burbulla, L. F., Kieper, N., Maurer, B., Waak, J., Wolburg, H., Gizatullina, Z. Z., Gellerich, F. N., Woitalla, D., et al. (2010) · PLoS ONE · doi:10.1371/journal.pone.0009367 · Cited by 381
“In contrast, the usage of GFP-WIPI-1 does not lead to such protein aggregations [25].”
BackgroundMethodSahani, M. H., Itakura, E., & Mizushima, N. (2014) · Autophagy · doi:10.4161/auto.27344 · Cited by 348
“1A).(8,20,21) LC3 conversion from LC3-I to LC3-II and inhibition of MTORC1 (dephosphorylation of RPS6KB/p70 S6 kinase and EIF4EBP1/4E-BP1) were simultaneously observed.”
Self-citationYang, B., Ding, L. S., Yao, H., Chen, Y., & Shi, J. (2020) · Advanced Materials · doi:10.1002/adma.201907152 · Cited by 363
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
“after lipidation becomes LC3-II, is inserted into the inner and outer membranes of the AP and is eventually degraded.(34)”
A rational mechanism for combination treatment of Huntington's disease using lithium and rapamycin
Sarkar, S., Krishna, G., Imarisio, S., Saiki, S., O’Kane, C. J., & Rubinsztein, D. C. (2007) · Human Molecular Genetics · doi:10.1093/hmg/ddm294 · Cited by 353
Zhu, K., Dunner, K., & McConkey, D. J. (2010) · Oncogene · doi:10.1038/onc.2009.343 · Cited by 338
“Accumulation of LC3II in the presence of lysosomal protease inhibitors is indicative of enhancement of autophagic flux (Mizushima and Yoshimori, 2007).”
BackgroundAutophagy is cytoprotective during cisplatin injury of renal proximal tubular cells
Periyasamy‐Thandavan, S., Jiang, M., Wei, Q., Smith, R., Yin, X., & Dong, Z. (2008) · Kidney International · doi:10.1038/ki.2008.214 · Cited by 321
“Together, these results have demonstrated evidence for the occurrence of autophagy during cisplatin-induced renal cell injury.”
BackgroundA Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1
Wang, J., Whiteman, M. W., Lian, H., Wang, G., Singh, A. K., Huang, D., & Denmark, T. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.026013 · Cited by 323
Defining and measuring autophagosome flux—concept and reality
Loos, B., du Toit, A., & Hofmeyr, J. S. (2014) · Autophagy · doi:10.4161/15548627.2014.973338 · Cited by 324
“Immunoblotting for endogenous LC3 is one of the crucial functional reporter assays that can be used to reliably monitor autophagosome synthesis or degradation, as LC3 associates with the lumenal and cytosolic surface of autophagosomes, with a degree of recycling taking place from the cytosolic surface.(9) However, wes…”
BackgroundKim, R. H., Coates, J. M., Bowles, T. L., McNerney, G. P., Sutcliffe-Goulden, J., Jung, J. U., Gandour‐Edwards, R. F., Chuang, F. Y. S., Bold, R. J., & Kung, H.-J. (2009) · Cancer Research · doi:10.1158/0008-5472.can-08-3157 · Cited by 321
“Increase in total autophagic flux was confirmed with chloroquine (30), an autophagy inhibitor that disrupts lysosomal function”
BackgroundWhy 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 312
Autophagy in ovary and polycystic ovary syndrome: role, dispute and future perspective
Kumariya, S., Ubba, V., Jha, R. K., & Gayen, J. R. (2021) · Autophagy · doi:10.1080/15548627.2021.1938914 · Cited by 306
Proline Metabolism and Microenvironmental Stress
Phang, J. M., Liu, W., & Zabirnyk, O. (2010) · Annual Review of Nutrition · doi:10.1146/annurev.nutr.012809.104638 · Cited by 303
“By contrast, the oxLDL induction of the conversion of LC3-I to LC3-II, hallmarks for the onset of autophagy (77), was markedly decreased by POX knockdown.”
Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy
Sinha, R. A., You, S.-H., Zhou, J., Siddique, M. M., Bay, B.-H., Zhu, X., Privalsky, M. L., Cheng, S.-Y., Stevens, R. D., Summers, S. A., et al. (2012) · Journal of Clinical Investigation · doi:10.1172/jci60580 · Cited by 303
Rodríguez-Navarro, J. A., Rodríguez, L., Casarejos, M. J., Solano, R. M., Gómez, A., Perucho, J., Cuervo, A. M., García de Yébenes, J., & Mena, M. A. (2010) · Neurobiology of Disease · doi:10.1016/j.nbd.2010.05.014 · Cited by 298
“However, LC3 II/LC3 I as well as LC3 II/β-actin ratio (Mizushima and Yoshimori, 2007), was increased after 2.”
InfluentialHigh-Mobility Group Box 1 Is Essential for Mitochondrial Quality Control
Tang, D., Kang, R., Livesey, K. M., Kroemer, G., Billiar, T. R., Houten, B. V., Zeh, H. J., & Lotze, M. T. (2011) · Cell Metabolism · doi:10.1016/j.cmet.2011.04.008 · Cited by 303
“, 2005), or blocking lysosomal degradation by pepstatin and E64D (lysosomal protease inhibitors) (Mizushima and Yoshimori, 2007) increased rotenone-mediated”
Li, L., Chen, Y., & Gibson, S. B. (2012) · Cellular Signalling · doi:10.1016/j.cellsig.2012.09.020 · Cited by 301
The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1
Vicencio, J. M., Ortiz, C., Criollo, A., Jones, A. W. E., Kepp, O., Galluzzi, L., Joza, N., Vitale, I., Morselli, E., Tailler, M., et al. (2009) · Cell Death & Differentiation · doi:10.1038/cdd.2009.34 · Cited by 289
“The redistribution of GFP-LC3 to dots or the accumulation of LC3-II may be a sign of an enhanced formation of autophagosomes or a reduced removal of autophagosomes.(33,34) To discriminate between these two possibilities, we blocked the fusion between autophagosomes and lysosomes (with bafilomycin A1) and/or inhibited…”
Campbell, G. R., & Spector, S. A. (2012) · PLoS Pathogens · doi:10.1371/journal.ppat.1002689 · Cited by 289
“The accumulation of LC3B-II was increased in the presence of the lysosomal protease inhibitor pepstatin A regardless of infection status (Figure 2B), indicative of autophagic flux [33].”
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 294
“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).”
Shehata, M., Matsumura, H., Okubo-Suzuki, R., Ohkawa, N., & Inokuchi, K. (2012) · The Journal of Neuroscience · doi:10.1523/jneurosci.4533-11.2012 · Cited by 285
“Monitoring autophagic flux is recommended, because the increase in the LC3-II/LC3-I ratio may be attributable to either the induction of AP formation or the blockade of AP–lysosomal fusion (Mizushima and Yoshimori, 2007).”
BackgroundTranscription Factor GATA4 Inhibits Doxorubicin-induced Autophagy and Cardiomyocyte Death
Kobayashi, S., Volden, P. A., Timm, D. J., Mao, K., Xu, X., & Liang, Q. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.070037 · Cited by 288
Kang, R., Tang, D., Schapiro, N. E., Livesey, K. M., Farkas, A. M., Loughran, P., Bierhaus, A., Lotze, M. T., & Zeh, H. J. (2009) · Cell Death and Differentiation · doi:10.1038/cdd.2009.149 · Cited by 340
“method for detecting autophagy is measuring enhanced degradation of p62 (SQSTM1/sequestosome), a long-lived scaffolding protein involved in the transport of ubiquitinated proteins destined for proteosomal digestion.(22,23) Indeed, knockdown of RAGE limited degradation of p62 induced by chemotherapeutic agents (Figure…”
MethodChoi, K. S. (2012) · Experimental & Molecular Medicine · doi:10.3858/emm.2012.44.2.033 · Cited by 291
“…of the regulatory mechanisms governing the early steps of autophagy, including autophagosome formation (initiation, nucleation and elongation) (Mizushima and Yoshimori, 2007), those governing the later steps of autophagy, such as autophagosomal maturation and lysosomal degradation, are less…”
BackgroundInfluentialAntagonism of Beclin 1‐dependent autophagy by BCL‐2 at the endoplasmic reticulum requires NAF‐1
Chang, N. C., Nguyen, M., Germain, M., & Shore, G. C. (2009) · The EMBO Journal · doi:10.1038/emboj.2009.369 · Cited by 292
“Thus, cell lysates were analysed by immunoblot (Swerdlow et al, 2008) for conversion of the cytoplasmic form of microtubule-associated protein 1 light chain 3 (LC3 I form) to LC3 II, which is widely used to assay autophagosome accumulation (Mizushima and Yoshimori, 2007; Figure 3C).”
BackgroundMethodRole of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy
Pattingre, S., Bauvy, C., Carpentier, S., Levade, T., Levine, B., & Codogno, P. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m805920200 · Cited by 290
Cao, Y., Chen, J., Ren, G., Zhang, Y., Tan, X., & Yang, L. (2019) · Nutrients · doi:10.3390/nu11112794 · Cited by 281
“Therefore, it is critical to explore the amount of LC3II delivered to lysosomes by comparing LC3II and p62 levels with or without the lysosomal protease inhibitors, including chloroquine or bafilomycin A1 [17].”
BackgroundAutophagy regulates selective HMGB1 release in tumor cells that are destined to die
Thorburn, J., Horita, H., Redzic, J., Hansen, K., Frankel, A. E., & Thorburn, A. (2009) · Cell Death & Differentiation · doi:10.1038/cdd.2008.143 · Cited by 268
“Autophagic flux assays were performed by western blotting for LC3-II formation after treatment with DTEGF in the presence or absence of lysosomal protease inhibitors as recommended by Mizushima and Yoshimori (25), by monitoring the processing of a GST-tagged version of the autophagy cargo protein betaine homocysteine…”
MethodControl of autophagy initiation by phosphoinositide 3‐phosphatase jumpy
Vergne, I., Roberts, E., Elmaoued, R. A., Tosch, V., Delgado, M. A., Proikas‐Cezanne, T., Laporte, J., & Deretic, V. (2009) · The EMBO Journal · doi:10.1038/emboj.2009.159 · Cited by 270
“To discern whether LC3-II increase following Jumpy or MTMR6 knock-downs was due to autophagy stimulation or inhibition of LC3-II degradation, Bafilomycin A1 (BafA1) and lysosomal protease inhibitors were added during the 30-min treatment (Mizushima and Yoshimori, 2007).”
Golgi-resident Small GTPase Rab33B Interacts with Atg16L and Modulates Autophagosome Formation
Itoh, T., Fujita, N., Kanno, E., Yamamoto, A., Yoshimori, T., & Fukuda, M. (2008) · Molecular Biology of the Cell · doi:10.1091/mbc.e07-12-1231 · Cited by 283
“To determine whether the observed effect was caused by upregulation of autophagosome formation or blockage of autophagic degradation (Mizushima and Yoshimori, 2007), accumulation of LC3-II in the presence of H /K ionophore nigericin was investigated.”
BackgroundSelf-citationAutophagy is involved in influenza A virus replication
Zhou, Z., Jiang, X., Liu, D., Fan, Z., Hu, X., Yan, J., Wang, M., & Gao, G. (2009) · Autophagy · doi:10.4161/auto.5.3.7406 · Cited by 275
Shimizu, S., Takehara, T., Hikita, H., Kodama, T., Tsunematsu, H., Miyagi, T., Hosui, A., Ishida, H., Tatsumi, T., Kanto, T., et al. (2011) · International Journal of Cancer · doi:10.1002/ijc.26374 · Cited by 269
“During the progress of autophagy, the cytoplasmic form LC3-I is converted to the membrane-bound lipidated form LC3-II which is detected by a mobility shift on electrophoresis.(15) When Huh7 cells were treated with 10 lM sorafenib, LC3 conversion was observed as early as 1 hr after the treatment and gradually increased…”
BackgroundSelf-citationAutophagy contributes to resistance of tumor cells to ionizing radiation
Chaachouay, H., Ohneseit, P., Toulany, M., Kehlbach, R., Multhoff, G., & Rodemann, H. P. (2011) · Radiotherapy and Oncology · doi:10.1016/j.radonc.2011.06.002 · Cited by 270
“of autophagosomes [16,17].”
Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites
Zoppino, F. C. M., Militello, R. D., Slavin, I., Álvarez, C., & Colombo, M. I. (2010) · Traffic · doi:10.1111/j.1600-0854.2010.01086.x · Cited by 265
“Therefore, autophagy can be monitored by the conversion of GFP-LC3-I to GFP-LC3-II, which can be detected by immunoblotting as LC3-II migrates faster than GFP-LC3-I during SDS–PAGE (42).”
BackgroundFilomeni, G., Graziani, I., Zio, D. D., Dini, L., Centonze, D., Rotilio, G., & Ciriolo, M. R. (2010) · Neurobiology of Aging · doi:10.1016/j.neurobiolaging.2010.05.021 · Cited by 260
The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy
Michiorri, S., Gelmetti, V, Giarda, E., Lombardi, F., Romano, F., Marongiu, R., Nerini-Molteni, S., Sale, P., Vago, R., Arena, G., et al. (2010) · Cell Death & Differentiation · doi:10.1038/cdd.2009.200 · Cited by 260
Kuo, T.-C., Chen, C., Baron, D. M., Önder, T. T., Loewer, S., Almeida, S., Weismann, C. M., Xu, P., Houghton, J., Gao, F., et al. (2011) · Nature Cell Biology · doi:10.1038/ncb2332 · Cited by 264
Metadata
- Authors
- Mizushima, N., & Yoshimori, T.
- Affiliations
- Tokyo Medical and Dental University (JP)
- Venue
- Autophagy
- Publisher
- Informa UK Limited
- Published
- 2007-11-26
- Volume
- 3
- Issue
- 6
- Pages
- 542-545
- ISSN
- 1554-8627 (ISSN-L)
- 1554-8635
- OpenAlex
- W1963521777
- Semantic Scholar
- ab5143ed104a787ff2e14d915df9a12ce5f5871a
- Cited by
- 2,595
- References
- 21
- Citations by year
- 2026: 62 · 2025: 114 · 2024: 152 · 2023: 141 · 2022: 176 · 2021: 164 · 2020: 173 · 2019: 155 · 2018: 177 · 2017: 162 · 2016: 167 · 2015: 179 · 2014: 137 · 2013: 165 · 2012: 120
- FWCI
- 52.76
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc
- Locations
- Autophagy (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- Kato Memorial Bioscience Foundation
- Toray Science Foundation
- Japan Society for the Promotion of Science
- 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, autophagic flux
- MeSH terms
- Animals, Autophagy, Humans, Lysosomes, Microtubule-Associated Proteins, Phosphatidylethanolamines, Sensitivity and Specificity, Immunoblotting, PC12 Cells, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, Rats
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