Fig. 3

PKG^RQ-induced oxidative stress, apoptosis, and reduced proliferation in human SMCs. Human aortic SMCs were infected with adenovirus encoding green fluorescent protein (control, in black and white), wild-type PKG1 (PKG1^WT, in b gray), PKG1^RQ (in red), or NOX4 (in purple), as indicated. (a, b) Similar amounts of PKG1^WT or PKG1^RQ were expressed (a). PKG activity was assessed in the absence and presence of cGMP following endogenous VASP phosphorylation in cells (a) or using a synthetic peptide (b). c, d Relative mRNA expression in SMCs expressing PKG1^RQ was normalized to phosphoglycerokinase-1 mRNA and compared to cells infected with control virus; some cells (in d) were treated with TGF-β for 24 h (gene names as in Fig. 2a, b). e, f NADPH oxidase activity and H₂O₂ production were measured in SMCs infected with control virus or virus encoding shRNA specific for NOX4 (NOX4 mRNA reduction by the shRNA is shown in Supplemental Fig. 4c). g H₂O₂ production was measured in cells infected with virus encoding PKG1^RQ or NOX4, and some cells were treated with the NOX1/4 inhibitor GKT137831 (GKT). h JNK activation was assessed in SMCs treated with vehicle or TGF-β. i–k DNA oxidation was assessed by immunofluorescence staining for 8-OH-deoxyguanosine (i: pink nuclei; DNA was counterstained with Hoechst 33342). Some cells were treated with GKT137831 or with the PKG inhibitor DT2. l, m SMC proliferation was assessed by Br-deoxyuridine (BrdU) uptake into S-phase nuclei, with some cells treated with GKT137831 or DT2. n, o Apoptosis was assessed by immunofluorescence staining for cleaved caspase-3 of cells cultured in 0.5% FBS. Graphs show means ± SEM of three (e, j), four (b, l, o), five (d, f, k, m, n), or six (c, g) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 for the comparison between PKG1^RQ- or NOX4-expressing cells versus control cells under the same condition (panel c by one-sample t-test, panels j–o by one-way ANOVA, and panels a and d–g, by two-way ANOVA). Source data are provided as a Source Data file