Figure 4.

HIF-1α mediates NOX4 expression. (A–C) Pulmonary artery smooth-muscle cells (PASMCs) were transfected with vectors coding for HIF-1α or with vectors coding for shRNA against HIF-1α (siH1I, siH1II) or for control shRNA (siCtr) and exposed to hypoxia for 4 h. (A) Western blot analyses were performed using antibodies against NOX4 or HIF-1α. Actin was used as loading control. (B) Northern blots were performed using specific probes for NOX4 or 18S. Data represent % change of NOX4 protein (A) or NOX4 mRNA levels (B) versus the appropriate normoxic control set to 100% (n = 3, *p < 0.05 vs. control; ^#p < 0.05 vs. hypoxic control). (C) mRNA levels for NOX4 and PAI-1 were determined in HIF-1α–overexpressing cells by real-time PCR using primers specifically amplifying NOX4, PAI-1, or actin fragments. Quantification was performed by ΔCT calculation. NOX4 or PAI-1 mRNA levels were normalized to actin levels. Control cells (Ctr, siCtr) were set to 100%, and the relative change in HIF-1α–overexpressing cells is displayed (n = 3; *p < 0.05 vs. control). (D) HepG2 cells were exposed to hypoxia for 3 h. Chromatin immunoprecipitation (ChIP) was performed with an antibody against HIF-1α. Real-time PCR was performed on the precipitates using primers for the NOX4 promoter (black) or the PAI-1 promoter (gray) as positive control or for the third intron of β-actin lacking an HRE as negative control (dark gray, neg. Ctr). For background calculation, ChIP without antibody was performed. Quantification is shown in promille to chromatin input for all samples after background subtraction (n = 3, *p < 0.05 vs. control).