2.3. Autophagy Flux Assay

Autophagy flux assay is one of the gold standard technics developed to assess autophagy [22]. Briefly, this technic relies on LC3, a cytosolic protein (LC3-I) that, upon autophagy induction, is conjugated to phosphatidylethanolamine (LC3-II). This conversion, described to be necessary for autophagosome formation, is used to measure the induction of autophagy in the presence of lysosomal inhibitors to prevent LC3-II degradation by hydrolases. The autophagy flux assay was therefore estimated by measuring the amount of LC3-II detected by Western blot in the presence or absence of a lysosomal inhibitor. The more the LC3-II level increases in the presence of the inhibitor, the higher the autophagy flux is supposed to be. Therefore, when indicated, cells were treated for the indicated times and media in the presence or absence of 10 µM chloroquine (CQ) (#C6628, Sigma-Aldrich) prior to proceeding to the protein extraction and Western blots analysis directed against the LC3B protein and tubulin as a loading control. Quantifications of LC3-II/tubulin ratios were then normalized to the ratio corresponding to the time point showing the highest autophagy flux (to arbitrarily define a 100% autophagy flux induction) according to the treatment considered.