FIG. 1.

(A) Schematic structures of HIF-1α and GAL4-H1α constructs used in this study. (B) Effects of hypoxia and hypoxia mimics on the transactivation activity of GH1α740-826. HeLa cells were cotransfected with GH1α740-826 (2.5 μg), pFR-luc reporter (2.5 μg), and pCMVLacZ (0.5 μg). Twenty-four hours later the transfected cells were trypsinized. Fifty percent of the cells were split equally into 12-well culture plates for luciferase assays (top), and the other 50% of the cells were split into 60-mm-diameter dishes to extract proteins for Western blotting with monoclonal antibody against GAL4-DBD (bottom). The cells were cultured under either normoxic (Nmx) or hypoxic (Hpx) (0.5% O₂) conditions or with desferrioxamine (Dfx) (130 μM)or cobalt chloride (Cbt) (75 μM), respectively, for 16 h before harvest. (C) Response of endogenous HIF-1 to hypoxia and hypoxic mimics in Hep3B cells. Hep3B cells were transfected with a pEpo-luc reporter (5 μg) and pCMVLacZ (0.5 μg), and the transfected cells were treated as described in panel B. The protein levels of HIF-1α in whole-cell lysates were detected with anti-HIF-1α monoclonal antibody. The luciferase activity was corrected by β-galactosidase activity. RLU, relative luciferase activity.