Hypoxia stimulation (3% O2) 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.
Fu et al. BMC Cell Biology 2010 11:66 doi:10.1186/1471-2121-11-66