Figure 1.

Hypoxia increases NOX4 expression in vitro. (A–C) Pulmonary artery smooth-muscle cells (PASMC) were stimulated for different time periods by hypoxia (1% O₂). (A) Northern blot analyses were performed using a specific probe for NOX4; 18S staining served as loading control. Data represent % change of NOX4 mRNA levels versus normoxic control (100%; n = 3, *p < 0.05 vs. control). (B) Real-time PCR was performed with primers amplifying cDNA fragments specific for NOX4, PAI-1, or actin. Quantification was performed by ΔCT calculation. NOX4 and PAI-1 mRNA levels were normalized to actin levels. Normoxic control (0) was set to 100% (n = 3; *p < 0.05 vs. control). (C) NOX4 and HIF-1α protein levels were determined by Western blot analyses. Actin levels served as loading control. Data represent % change of NOX4 protein levels versus normoxic control (100%; n = 5, *p < 0.05 vs. control). (D) HEK293 cells were cotransfected with a plasmid encoding GFP-NOX4 and plasmids encoding shRNA against NOX4 (siN4I) or control shRNA (siCtr). Western blot analyses were performed with antibodies against NOX4 and GFP. Staining with an antibody against actin served as loading control. GFP or NOX4 protein levels in siCtr-expressing cells were set to 100%. Data represent % decrease of GFP or NOX4 protein levels compared with control (n = 3; *p < 0.05 vs. control). (E) PASMCs were transfected with vectors encoding two different shRNAs against NOX4 (siN4I, siN4II) or control shRNA (siCtr) and exposed to hypoxia for 4 h. Western blot analyses were performed with an antibody against NOX4. Actin staining served as loading control. NOX4 protein levels in hypoxic siCtr-expressing cells were set to 100% (n = 3; *p < 0.05 vs. control).