Full-length MYLK promoter (2.5 kb) was constructed into upstream of firefly luciferase gene in pGL3-basic. Human pulmonary artery ECs were transfected by full-length MYLK promoter construct along with Renilla luciferase reporter phRL-TK as normalization control vector. (A) nmMYLK promoter constructs transfected ECs were exposed to LPS (100 ng/ml), TNF-α (10 ng/ml), or 18% CS for 1, 2, 4, 8, and 24 h, and luciferase activity measured using the Dual Luciferase Assay System (Promega). The bar graph represents normalized relative luciferase activity by vehicle-treated control (*P<0.05 vs. control, n=4 each group). Compared with controls, LPS, TNF-α (10 ng/ml), or 18% CS all significantly increased nmMYLK promoter activities in time-dependent manner. (B) nmMYLK promoter constructs transfected ECs were exposed to the combination of LPS (100 ng/ml) and 18% CS for 1, 2, 4, 8, and 24 h, and luciferase activity measured. Compared with controls, the combination of LPS and 18% CS significantly increased nmMYLK promoter activities at all time points (*P<0.05 vs. control, n=6 each group). (C) nmMYLK promoter activities were significantly increased by HIF-1/2α. Human ECs were co-transfected with nmMYLK promoter reporter with scramble siRNA (siCtrl), siRNA for HIF-1α or HIF-2α, then treated with vehicle or HIF prolylhydroxylase (PHD) inhibitor FG-4592 (100 mM) for 4, 24, and 48 h, and luciferase activity was measured. FG-4592 significantly increased MYLK promoter activities (**P<0.01 vs. vehicle). Effects of FG-4592 were significantly attenuated by siHIF-1α at 4 and 24 h, decreased by HIF2α at 24 and 48 h (*P<0.01 vs. FG-4592 with siCtrl) (n=4 each).