HIF-1α and NOX4 modulate ROS levels under normoxia and hypoxia. (A) Pulmonary artery smooth-muscle cells (PASMCs) were transfected with plasmids coding for NOX4 or for HIF-1α and were cotransfected with shRNA against NOX4 (siN4I) or with control shRNA (siCtr). ROS levels were evaluated by DHE fluorescence. ROS levels of cells transfected with control vectors were set to 100% (n = 3; *p < 0.05 vs. control, #p < 0.05 vs. HIF-1α). (B) PASMCs were transfected with a plasmid coding for NOX4 and were exposed to hypoxia for 30 min (0.5 h hypoxia), or with plasmids coding for shRNA against NOX4 (siN4I, siN4II), HIF-1α (siH1I, siH1II) or with control shRNA (siCtr) and exposed to hypoxia for 4 h. ROS levels were evaluated by DHE fluorescence thereafter. ROS levels of cells transfected with control vectors under normoxic conditions were set to 100% (n = 3; *p < 0.05 vs. normoxic controls, #p < 0.05 vs. hypoxic control). (C) PASMCs were transfected with plasmids coding for either shRNA against NOX4 (siN4I), HIF-1α (siH1I), or control shRNA (siCtr). Cells were exposed to hypoxia for 4 h, and ROS levels were measured by EPR using the spin-trap CMH. ROS levels of hypoxic control cells (siCtr) were set to 100% (n = 3; *p < 0.05 vs. hypoxic control).