Fig. 2

Hypoxia promotes RASSF1A protein stability and HIF1α regulates its transcription. a Human PASMCs were pre-treated with NAC (N-acetyl cysteine) at indicated concentrations for 1 h or b transfected with NOX1 siRNA for 48 h or c pre-treated with Gö6976 at the indicated concentrations for 1 h. a–c Treatments were followed by 15 min hypoxia exposure, a–c, upper western blotting and a–c lower quantification of RASSF1A expression. d, e HEK 293 cells were transfected with RASSF1A-FLAG or empty vector (EV), and exposed to hypoxia for 30 min, d without or e with pre-treatment of Gö6976 for 1 h, followed by phosphoserine IP and RASSF1A blotting. f Human PASMCs were transfected with PKCα siRNA (si-PRKCA) for 48 h, followed by 15 min hypoxia exposure, western blotting, and f, right densitometric quantification of RASSF1A. g, h HEK293 cells were transfected with plasmids as indicated, treated with 30 μg/ml cycloheximide (CHX) without g or with h MG132 pretreatment, followed by hypoxia exposure for 1 h and 3 h and western blotting. g, h, lower Densitometrical quantified data of % of RASSF1A remaining. i Human PASMCs were transfected with HIF1α siRNA (si-HIF1A) or HIF2α siRNA (si-HIF2A) or both, followed by 24 h hypoxia exposure, western blotting for indicated proteins i, upper and densitometric quantification i, lower of RASSF1A. j, left In silico analysis of HIF-1 binding sites (HBS) in human RASSF1A promoter. j, right Human PASMCs were exposed to hypoxia for 24 h, followed by ChIP with anti-HIF1α (HIF1A) and real time PCR with primers spanning the putative HBS sites in RASSF1A promoter. k HEK293 cells were transfected with indicated luciferase promoter plasmids, followed by 24 h hypoxia exposure and luciferase activity measurement. RLU relative luciferase units. *P < 0.05, **P < 0.01, ***P < 0.001 compared to b, f, i si-Control (Hypoxia), a, c, h Vehicle, g RASSF1A-FLAG at same time point, j IgG or k EV, one-way ANOVA followed by SNK multiple comparison test. g ^§§§P < 0.001 compared to RASSF1A-FLAG (S203A), two-way ANOVA. Data represent mean ± s.e.m. n = 3 independent experiments from 3 biological replicates for human PASMCs and n = 3 independent experiments for HEK cells