Modulation of A23187- and BAPTA-am-dependent HIF-1α induction by Act D and CHX. HepG2 cells were pretreated with Act D (5 μg/mL) or CHX (10 μg/mL) for 30 minutes, then stimulated with A23187 (5 μM) or BAPTA-am (5 μM) under normoxia (16% O2) or hypoxia (8% O2) for 4 hours. HIF-1α protein was detected by Western blot analysis. (A,C) The expression of HIF-1α under hypoxia was set to 100%. Values are means ± SEM of 3 independent culture experiments. Statistics, Student t test for paired values: *P ≤ 0.05 versus controls at the same pO2; **P ≤ .05 versus A23187 treatment group at the same pO2. (B,D) Representative Western blot. Protein (100 μg) from the whole-cell extract was subjected to Western analysis with an antibody against HIF-1α or β-actin. Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry.
