Fig. 2

Hypoxia induces autophagy in cancer cells. a: LC3-GFP distribution by confocal microscopy. A549 cells were incubated in normoxia or hypoxia for 8 h. GFP-LC3 showed diffuse intracellular localization under normoxic conditions. Membrane translocation (punctate localization) indicative of autophagy was observed in hypoxic cells (magnification x40). White arrows, punctate GFP-LC3. b: Quantification of GFP-LC3 translocation in transiently transfected normoxic and hypoxic cells. The number of GFP-LC3 spots per cell was counted. c: A549 cells were incubated under normoxia or hypoxia for 2, 4, 6 or 8 h. Western blots showed LC3B-I to LC3B-II conversion in A549 cells incubated in 1 % hypoxia compared with normoxia. Expression of the p62 marker of autophagy flux decreased in A549 cells incubated at 1 % hypoxia compared with normoxia. d, e: Intensity of Western blots measured by Image J. Graphs represents 6 independent experiments. f: Electron micrographs of A549 cell ultrastructure after normoxia or hypoxia for 8 h. Black arrows, double-membraned autophagic vacuoles (magnification x20,000). g: Quantification of autophagic vacuoles in normoxic and hypoxic cells. The number of autophagic vacuoles per cell was counted. N, normoxia; H, hypoxia. Data are mean ± SEM, *p < 0.05