title: Measuring autophagy in stressed cells.
abstract: Macro-autophagy is a major catabolic process in the cell used to degrade protein aggregates, dysfunctional organelles and intracellular pathogens that would otherwise become toxic. Autophagy also generates energy and metabolites for the cell through recycling of degraded autophagosomal cargo, which can be particularly important for cell viability under stress. The significance of changes in the rates of autophagic flux for cellular function and disease is being increasingly appreciated, and interest in measuring autophagy in different experimental systems is growing accordingly. Here, we describe key methodologies used in the field to measure autophagic flux, including monitoring LC3 processing by western blot, fluorescent cell staining, and flow cytometry, in addition to changes in the levels or posttranslational modifications of other autophagy markers, such as p62/Sqstm1 and the Atg5-Atg12 conjugate. We also describe what cellular stresses may be used to induce autophagy and how to control for changes in the rates of autophagic flux as opposed to inhibition of flux. Finally, we detail available techniques to monitor autophagy in vivo.
authors: The Ben May Department for Cancer Research, The Gordon Center for Integrative Sciences, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA.
authors: Marina N
authors: MN
authors: Sharifi
authors: Erin E
authors: EE
authors: Mowers
authors: Lauren E
authors: LE
authors: Drake
authors: Kay F
authors: KF
authors: Macleod
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071187:Autophagy-Related Protein 5; D015153:Blotting, Western; D006801:Humans; D007150:Immunohistochemistry; D018942:Macrolides / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D000071456:Sequestosome-1 Protein
chemical_list: C502778:ATG5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000071187:Autophagy-Related Protein 5; D018942:Macrolides; D008869:Microtubule-Associated Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; C040929:bafilomycin A1