Western blot analysis of HIF-1α in extracts from human osteosarcoma cells (U2OS) treated with various mitochondrial inhibitors. Cells were cultured for 4 hours in normoxia (NOX) or hypoxia (HOX) with or without inhibitors of the ETC. The transcription factor SP-1, which is not responsive to hypoxia, served as a control for uniform loading and transfer. (A) Cells were cultured in intermediate hypoxia (3% O₂) on conventional polystyrene dishes. (B) Cells were cultured in intermediate hypoxia (3% O₂) on gas-permeable dishes. (C) Cells were incubated with or without antimycin A (1 μg/mL) or rotenone (1 μM) for 4 hours under strong hypoxic conditions (0.1% O₂) on conventional cell culture supports.