Figure 11.

siNOX4 blocks TRAIL‐inducible NO signaling. A, TRAIL‐inducible (10 ng/mL) NOX4 mRNA expression is inhibited with l‐NAME. B, TRAIL‐induced eNOS phosphorylation Ser‐1177 at 15 minutes is blocked with siNOX4 (200 nmol/L). Western blotting for p‐eNOS ^(S1177) and α‐tubulin showing unbiased loading. The intensity of eNOS phosphorylation at Ser‐1177 compared to α‐tubulin was determined by densitometry (AU) (C) Left panel, TRAIL‐inducible (10 ng/mL) intracellular NO is inhibited by siNOX4 compared to the control siC. DAF‐FM fluorescence in green measuring NO levels; nuclei were stained with Hoechst (blue). Right panel, quantification of intracellular NO by HMEC‐1 cells as described in the Methods section. Magnification bar=100 μm. Data represent the combined results of at least 3 independent experiments. Results are expressed as mean±SEM; 1‐way ANOVA with Bonferroni comparison; *P<0.05, **P<0.01, and ****P<0.0001. AU indicates arbitrary units; DAF‐FM, 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein diacetate; e‐NOS, endothelial nitric oxide synthase; HMEC‐1, human microvascular endothelial cell‐1; l‐NAME, l‐NG‐nitroarginine methyl ester; NO, nitric oxide; NOX4, NADPH oxidase 4; NT, no treatment; TRAIL, tumor necrosis factor–related apoptosis‐inducing ligand.