Abstract
Autophagy and ER stress are most often studied employing a Western blotting approach to the measurement of autophagy by LC3B upregulation and the ER stress sensor signaling proteins PERK (protein kinase R-like endoplasmic reticulum kinase), IRE1, and ATF6 which initiate protein refolding and elongation of the ER until ER homeostasis is returned. If the misfolding of proteins is increased, then ER stress is maintained, and microautophagy of the ER or specifically reticulophagy occurs. However, LC3B, PERK, protein misfolding, and changes in ER mass (reticulophagy) can also be measured in a cell cycle-dependent manner by flow cytometry and the use of antibodies, protein misfolding, and ER tracking fluorescent probes.
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References
Ashford TP, Porter K (1962) Cytoplasmic components in hepatic cell lysosomes. J Cell Biol 12:198–202
Deter RL, Duve CD (1967) Influence of glucagon, an inducer of cellular autophagy, on some physical properties of rat liver lysosomes. J Cell Biol 33:437–449
Tooze S, Yoshimori T (2010) The origin of the autophagosomal membrane. Nat Cell Biol 12:831–835
Mehrpour M, Esclatine A, Beau I, Codogno P (2010) Overview of macroautophagy regulation in mammalian cells. Cell Res 20:748–762
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, **ao T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO 19:5720–5728
Barth S, Glick D, Macleod K (2010) Autophagy: assays and artifacts. J Pathol 221:117–124
Hansen TE, Johansen T (2011) Following autophagy step by step. BMC Biol 9:1–4
Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L, Amelio I, Andrews DW, others (2018) Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 25:486–541
Choi J, Yoon J, Won Y, Park B, Lee Y (2012) Chloroquine enhances the chemotherapeutic activity of 5-fluorouracil in a colon cancer cell line via cell cycle alteration. APMIS 120:597–604
Kapuy O, Vinod PK, Banhegyi G (2014) mTOR inhibition increases cell viability via autophagy induction during endoplasmic reticulum stress - an experimental and modeling study. FEBS Open Bio 4:704–713
Ganley I, Wong P, Gammo N, Jiang X (2011) Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell 42:731–743
Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, others (2006) Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 26:9220–9231
Bernales S, McDonald K, Walter P (2006) Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLoS Biol 4:e423
Yorimitsu T, Klionsky D (2007) Eating the endoplasmic reticulum: quality control by autophagy. Trends Cell Biol 17:279–285
Mandl J, Mészáros T, Bánhegyi G, Hunyady L, Csala M (2009) Endoplasmic reticulum: nutrient sensor in physiology and pathology. Trends Endocrinol Metabol 20:194–201
Popat A, Patel A, Warnes G (2019) A flow cytometric study of ER stress and autophagy. Cytometry A 95:672–682
Thomas S, Thurn KT, Biçaku E, Marchion DC, Münster N (2011) Addition of a histone deacetylase inhibitor redirects tamoxifen-treated breast cancer cells into apoptosis, which is opposed by the induction of autophagy. Breast Cancer Res Treat 130:437–447
Geng Y, Kohil L, Klocke BJ, Roth KA (2010) Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent. Neuro-Oncology 12:473–481
Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson S (2007) Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ 15:171–182
Shen D, Coleman J, Chan E, Nicholson T, Dai L, Sheppard P, Patton W (2011) Novel cell- and tissue-based assays for detecting misfolded and aggregated protein accumulation within aggresomes and inclusion bodies. Cell Biochem Biophys 60:173–185
Chikte S, Panchal N, G W. Use of LysoTracker dyes: a flow cytometric study of autophagy. Cytometry A 2014;85A:169–178
Warnes G (2015) Flow cytometric assays for the study of autophagy. Methods 82:21–28
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Popat, A., Patel, A.A., Warnes, G. (2022). Cell Cycle Analysis of ER Stress and Autophagy. In: Barcenilla, H., Diaz, D. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 2543. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2553-8_13
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DOI: https://doi.org/10.1007/978-1-0716-2553-8_13
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