Macroautophagy (hereafter referred to as autophagy), a central mechanism mediating the lysosomal degradation of cytoplasmic components, can be stimulated by a wide panel of adverse stimuli, including a panoply of anticancer agents. The central autophagic organelle is the autophagosome, a double membrane-bound vacuole that sequesters the cytoplasmic material destined to disposal. The ultimate destiny of the autophagosome is to fuse with a lysosome, resulting in the degradation of the autophagic cargo. In this setting, it is important to discriminate whether a particular stimulus actually promotes autophagy or it simply blocks the fusion of autophagosomes with lysosomes. To this aim, the methods that assess autophagy should assess not only the number of autophagosomes but also the so-called autophagic flux, that is, the clearance of the autophagy cargo from the lysosomal compartment. Here, we present a compendium of methods to assess the autophagic flux in cultured malignant cells. This approach should allow for the study of the intimate link between autophagy and oncometabolism in several experimental paradigms.
Keywords: Autophagy-related proteins; Electrophoresis; Fluorescence microscopy; Lysosome; Macroautophagy; Proteolysis.
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