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Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3

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Proteostasis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1449))

Abstract

Macroautophagy (called just autophagy hereafter) is an intracellular degradation machinery essential for cell survival under stress conditions and for the maintenance of cellular homeostasis. The hallmark of autophagy is the formation of double membrane vesicles that engulf cytoplasmic material. These vesicles, called autophagosomes, mature by fusion with endosomes and lysosomes that allows the degradation of the cargo. Autophagy is a dynamic process regulated at multiple steps. Assessment of autophagy is not trivial because the number autophagosomes might not necessarily reflect the real level of autophagic degradation, the so-called autophagic flux. Here, we describe an optimized protocol for the analysis of relevant parameters of autophagic flux using HeLa cells stably expressing EGFP-LC3. These cells are a convenient tool to determine the influence of the downregulation or overexpression of specific proteins in the autophagic flux as well as the analysis of autophagy-modulating compounds. Western blot analysis of relevant parameters, such as the levels of EGFP-LC3, free EGFP generated by autophagic degradation and endogenous LC3·I–II are analyzed in the presence and absence of the autophagic inhibitor chloroquine.

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Acknowledgments

This work was supported by grants BFU2012-32536 and BFU2015-64440-P from the Spanish Ministerio de Ciencia e Innovación. SMB is recipient of a predoctoral fellowship from Universidad Autónoma de Madrid. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).

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Authors and Affiliations

  1. Instituto de Investigaciones Biomédicas Alberto Sols, Arturo Duperier 4, Madrid, 28029, Spain

    Sandra Muñoz-Braceras & Ricardo Escalante

Authors
  1. Sandra Muñoz-Braceras
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  2. Ricardo Escalante
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Correspondence to Ricardo Escalante .

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Editors and Affiliations

  1. Computational and Experimental Biology Group Department of Health Promotion and Chronic Diseases, National Health Institute Dr. Ricardo Jorge, INSA, I.P., Lisboa, Portugal

    Rune Matthiesen

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Muñoz-Braceras, S., Escalante, R. (2016). Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_20

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  • DOI: https://doi.org/10.1007/978-1-4939-3756-1_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3754-7

  • Online ISBN: 978-1-4939-3756-1

  • eBook Packages: Springer Protocols

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