Figure 3.

Hypoxia-induced autophagy drives Cav-1 degradation in fibroblasts: Rescue with chloroquine, an autophagy inhibitor. (A) Hypoxia decreases Cav-1 levels in homotypic fibroblasts. Homotypic cultures of hTERT-fibroblasts were placed in hypoxia (0.5% O₂) or normoxia (21% O₂) for three days. Then, cells were fixed and stained with anti-Cav-1 (red) antibodies and DAPI nuclear stain (blue). Cav-1 staining (red only) is shown at left to better appreciate the hypoxia-induced Cav-1 degradation. Original magnification, 40x. (B) Hypoxia-induced Cav-1 downregulation correlates with the expression of autophagic markers. hTERT-fibroblasts were subjected to hypoxia (0.5% O₂, lower panels) or normoxia (21% O₂, upper panels) for 48 hours. Then, the cells were fixed and stained with antibodies against either Cav-1 (green) or the indicated autophagic markers (red). DAPI was used to stain nuclei (blue). Note that hypoxia induces Cav-1 downregulation, while promoting autophagy. Original magnification, 80x. (C) The autophagy inhibitor chloroquine rescues the hypoxia-induced downregulation of Cav-1. To block hypoxia-induced Cav-1 degradation, hTERT-fibroblasts were subjected to hypoxia in the presence of the autophagic inhibitor chloroquine (25 µM) or vehicle alone control (H₂O) for 24 hours. Then, cells were fixed and stained with anti-Cav-1 (green) antibodies and DAPI nuclear stain (blue). Note that chloroquine treatment prevents the hypoxia-induced degradation of Cav-1, as compared with control cells. Original magnification, 60x.