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. 2010 Jun 15;21(12):2087–2096. doi: 10.1091/mbc.E09-12-1003

Figure 3.

Figure 3.

Hypoxia increases NOX4 transcription. (A and B) Pulmonary artery smooth-muscle cells (PASMCs) were treated with actinomycin D (Act, 5 μM) or DMSO (Ctr) for 1 h and exposed to hypoxia for 4 h. (A) Northern blot analyses were performed using a specific probe for NOX4, 18S staining served as loading control. NOX4 mRNA levels in DMSO-treated cells under hypoxia (Ctr) were set to 100%. Data represent % change of NOX4 mRNA levels versus hypoxic control (n = 3, *p < 0.05 vs. hypoxic control). (B) Western blot analyses were performed using antibodies against NOX4 or HIF-1α. Actin served as loading control. Protein levels in DMSO-treated cells under hypoxia (Ctr) were set to 100%. Data represent % change of protein levels versus hypoxic control (n = 3, *p < 0.05 vs. hypoxic control). (C) HEK293 cells were cotransfected with luciferase constructs containing either the wild-type NOX4 promoter (NOX4-730) or the NOX4 promoter mutated at the hypoxia-responsive element (HRE; NOX4-730m). Cells were exposed to hypoxia (Hyp) or were cotransfected with a plasmid coding for HIF-1α. Luciferase activities under the respective control conditions (Ctr) for each reporter plasmid were set equal to 100%. Data represent % induction of luciferase activity (n = 3; *p < 0.05 vs. control).