Fig. 2
From: Aortic pathology from protein kinase G activation is prevented by an antioxidant vitamin B12 analog
Gene expression changes, Nox4 upregulation, oxidative stress, and increased MMP activity in Prkg1RQ/+ aortas. a, b Relative mRNA expression in aortas of 4-month-old PKG1RQ/+ mice compared to wild-type mice: smooth muscle α2-actin (Acta2); myosin heavy chain-11 (Myh11); transgelin (Tagln); myocardin (Myocd); TGF-β1 (Tgfb1); plasminogen activator inhibitor-1 (Serpine1); connective tissue growth factor (Ctgf); collagen3-α1 (Col3a1); elastin (Eln); lumican (Lum); decorin (Dcn); and NADPH oxidase-2 and -4 (Nox2, Nox4). Genes of interest were normalized to 18S rRNA and the mean ΔCt of WT mice was assigned a value of one (n = 3–4 M + 3–4 F mice per genotype). c H2O2 production of aortic SMCs isolated from WT (in blue) and PKGRQ/+ (in red) mice measured over time by Amplex Red fluorescence. d–g Oxidative stress markers measured in aorta and serum of 8- to 12-month-old WT and PKGRQ/+ mice. DNA oxidation was assessed by immunohistochemical staining of 8-OH-deoxyguanosine (d: arrows point to examples of brown nuclei counted as positive; scale bar 50 μm, n = 2 M + 3 F mice per genotype). Lipid peroxidation was assessed by measuring aorta malondialdehyde (MDA) content using thiobarbituric acid (e, n = 2 M + 5 F mice). Ascorbyl radical was measured in serum by electron paramagnetic resonance (f, n = 2 M + 5 F mice). Protein carbonyl groups were detected by OxyBlotTM after derivatization of aortic extracts with 2,4-dinitrophenylhydrazine (DNPH); non-derivatized extracts served as control and reprobing with a β-actin antibody showed equal loading (g, ImageJ quantification for n = 2 M + 2 F mice per genotype below). h JNK activation was assessed on western blots of aortic extracts using a phospho-specific antibody, and was normalized to β-actin (ImageJ quantification for n = 4 M + 6 F mice per genotype below). i, j Metalloproteinase (MMP) activity was assessed in aortic extracts by gelatin zymography (i, n = 1 M + 2 F mice per genotype) and measured using fluorescently labeled elastin as a substrate (j, n = 2 M + 4 F mice per genotype; conditioned medium from MDA-MB231 cells served as positive control). Graphs (a, g) show means ± SEM, other plots means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons by one-sample t-test (panels a, b, g), two-sided t-test (e, f, h, j), or Mann–Whitney test (d). Source data are provided as a Source Data file
