Detection of apoptosis by TUNEL: Staining. A549 cells were cultured on cover slides and transfected with different plasmids. Cells were allowed to recover in regular culture medium for 20 h after transfection, then exposed to normoxic or hypoxic atmosphere for 24 h with or without HEPES (25 mM) or the glycolytic inhibitor, 2-DG (5 mM) (n = 5 in each group). Only occasional normoxic cells were positive for TUNEL staining, (A). Hypoxia increased the amount of apoptosis of A549 cells, (D). Over-expression of HIF-1α further increased this pro-apoptotic effect of hypoxia, (F). However, siRNA suppression of HIF-1α expression rescued A549 cells from hypoxia-induced apoptosis, (C); but scrambled siRNA did not have this effect, (E). The pro-apoptotic effect of HIF-1α was also inhibited by the glycolytic inhibitor, 2-DG, (G). Increasing the buffering capacity of the culture medium with HEPES also inhibited the pro-apoptotic effect of HIF-1α, (H). Shown are representative photomicrographs from five independent experiments. Figure 9 was statistics of the five independent experiments. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES. Scale bar: 100 μm.