Sharifi Marina N, Mowers Erin E, Drake Lauren E, Macleod Kay F
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.
Methods Mol Biol. 2015;1292:129-50. doi: 10.1007/978-1-4939-2522-3_10.
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.
巨自噬是细胞内一种主要的分解代谢过程，用于降解蛋白质聚集体、功能失调的细胞器和细胞内病原体，否则这些物质会变得有毒。自噬还通过回收降解的自噬体货物为细胞产生能量和代谢物，这对于应激状态下的细胞存活尤为重要。自噬通量速率变化对细胞功能和疾病的重要性日益受到重视，相应地，在不同实验系统中测量自噬的兴趣也在增加。在此，我们描述了该领域用于测量自噬通量的关键方法，包括通过蛋白质印迹法监测LC3加工、荧光细胞染色和流式细胞术，此外还包括其他自噬标志物（如p62/Sqstm1和Atg5-Atg12共轭体）水平或翻译后修饰的变化。我们还描述了哪些细胞应激可用于诱导自噬，以及如何控制自噬通量速率的变化而非通量抑制。最后，我们详细介绍了用于在体内监测自噬的现有技术。
