FIG. 1.
Hypoxia induces autophagy in an HIF-dependent manner in normal and cancer cell lines. (A) Conversion of LC3-I to LC3-II in CCL39 cells subjected to 24 and 48 h of hypoxia (H 1%) compared to that of cells in normoxia (N). Total cellular extracts were analyzed by immunoblotting with antibodies against HIF-1α and LC3. The LC3-I and LC3-II bands were quantified, and autophagy was reported as a variation in the ratio of LC3-II/LC3-I for each condition. n.s., nonspecific. (B) Conversion of LC3-I to LC3-II in MCF7, LS174, and PC3 cells subjected to 24 and 48 h of hypoxia for the indicated times. Total cell extracts were analyzed by immunoblotting with antibodies against HIF-1α and LC3. (C) Accumulation of LC3-II in the presence of bafilomycin A1 (Bafilo. A1) in CCL39 cells. CCL39 cells were incubated in hypoxia (24 h) and treated with bafilomycin A1 (10 nM for 1 or 4 h) prior to the end of hypoxic treatment. Total cellular extracts were analyzed by immunoblotting with antibodies against LC3. β-Actin was used as a control. (D) MDC staining of PC3 cells. PC3 cells were treated for 48 h in normoxia or hypoxia in the absence (− bafilomycin A1) or presence (+ bafilomycin A1) of bafilomycin A1 (1 nM for 48 h), and during the last hour 0.1 μM MDC was added. The quantification of MDC-stained structures was performed using the analyze particles tool of the Image J software on an average of 100 cells per treatment. Baf. A1, bafilomycin A1. (E) Ablation of Beclin1 totally suppresses hypoxia-induced autophagy. The inset shows the ablation of Beclin1 with a shBeclin1 lentivirus. Cont., control. PC3 cells were infected with shBCN. Total cell extracts were analyzed by immunoblotting with antibodies against Beclin1. β-Actin was used as a control. For immunoblotting, PC3 control and shBCN cells were subjected to either normoxia or hypoxia (48 h). Total cell extracts were analyzed by immunoblotting with antibodies against LC3. β-Actin was used as a control. (F) Knockout of HIF-1α suppresses hypoxia-induced autophagy. MEFs derived from wild-type (HIF+/+) and HIF-1α knockout embryos (HIF−/−) were subjected to either normoxia or hypoxia (48 h). Total cell extracts were analyzed by immunoblotting with antibodies against HIF-1α and LC3. Tubulin was used as a control. (G) Suppression of HIF-2α totally represses hypoxia-induced autophagy. Total cell extracts of renal clear cell carcinoma 786-O cells not expressing pVHL (−) or expressing pVHL (+) in normoxia were analyzed by immunoblotting with antibodies against HIF-1α, HIF-2α, and LC3. Tubulin was used as a control.