Hypoxia stimulation (3% O
) induces expression and activation of HIF-1α in cultured HUASMCs. (A) Hypoxia (3% O2) induces HIF-1α mRNA expression. Total cellular RNA was isolated from control HUASMCs under normoxia or hypoxia-stimulated cells in the presence or absence of inhibitors. After reverse transcription, they were subjected to quantitative PCR analysis to determine HIF-1α mRNA level. Graph is representative of relative HIF-1α mRNA levels in the various conditions (n = 3 in each group). * indicates P < 0.05 vs control cells under normoxia. # P < 0.05 vs cells exposed to hypoxia for 24 h. PD98: PD98059. (B) Hypoxia (3% O2) increases HIF-1α protein levels. Western Blot detecting of HIF-1α protein expression in control HUASMCs under normoxia or cells exposed to hypoxia (3% O2) in the presence or absence of inhibitors. Representative Western blot (top) and values of HIF-1α production (mean ± SEM of 3 experiments, bottom). Results of HIF-1α protein production were obtained from densitometric analysis and expressed as ratio of HIF-1α/β-actin. * P < 0.05 vs control cells under normoxia. # P < 0.05 vs cells exposed to hypoxia for 24 h. PD98: PD98059. (C) Hypoxia increases HIF-1α DNA binding activity (EMSA results). Representative electrophoretic mobility shift assay (EMSA) showing protein binding to the HIF-1α oligonucleotide in nuclear extracts of HUASMCs after hypoxia stimulation in the presence or absence of inhibitors. Similar results were found in another two independent experiments.