TRAIL increases NOX4 expression. A, Three days ischemic Trail
−/− hindlimbs express less NOX4 mRNA compared to WT. Results are expressed as a ratio of ischemic vs control gastrocnemius muscles 3 days after hindlimb ischemia. NOX4 expression was normalized to 18S (WT, n=4; Trail
−/−, n=4). B, NOX4 mRNA expression is increased with TRAIL treatment. Serum arrested HMEC‐1 cells were treated with 10 ng/mL TRAIL for 24 hours followed by qPCR. TRAIL expression was normalized to GAPDH. C, Western blotting demonstrating that TRAIL (10 ng/mL) increases NOX4 protein expression over time in HMEC‐1 cells. The loading control is represented by α‐tubulin. The intensity of NOX4 expression compared to α‐tubulin was determined by densitometry (AU). D, NOX1 and (E) NOX2 mRNA expression is unchanged with 10 ng/mL TRAIL treatment. Expression was normalized to GAPDH. Unless stated, the data represent the combined results of at least 3 independent experiments. Results are expressed as mean±SEM; Mann–Whitney U‐test, t test, or 1‐way ANOVA with Bonferroni comparison; *P<0.05, **P<0.01, and ***P<0.001. AU indicates arbitrary units; HMEC‐1, human microvascular endothelial cell‐1; NOX4, NADPH oxidase 4; NT, no treatment; qPCR, quantitative polymerase chain reaction; TRAIL, tumor necrosis factor–related apoptosis‐inducing ligand; WT, wildtype.