Nox4 regulated CSE transcription through the HRI/eIF2α/ATF4 signaling module.
A, QPCR analyses of HRI and CSE mRNA expression in HUVEC after 48 h of treatment with siRNA targeted to HRI (siHRI) or control siRNA (siScram) together with 24-h Nox4 or β-gal overexpression as indicated. A.U., absorbance units. B, representative Western blot analyses of Nox4, total eIF2α, and phosphorylated eIF2α protein (Pi-eIF2α) and quantitative densitometric analyses of Pi-eIF2α, normalized to total eIF2α phosphorylation, after treatments as in A. C, representative Western blot analyses and quantitative densitometric analyses of ATF4 protein expression after treatments as in A. D and E, QPCR analyses of Nox4 and CSE mRNA expression in HUVEC after inhibition of heme biosynthesis with 1 mm 4,6-dioxoheptanoic acid (DA) (D) or inhibition of heme oxygenase-1 with 10 μm tin protoporphyrin (SnPP) (E) for 24 h. All data were normalized to β-actin mRNA and protein expression apart from B. n = 3 for mRNA analyses, n = 5 for protein analyses. *, p < 0.05; **, p < 0.01; ***; p < 0.001; ****, p < 0.0001.