Effects of SIRT5 silencing on TNF-α-mediated PAI-1 expression in primary human aortic endothelial cells. (A) In HAECs, SIRT5 protein expression is significantly reduced after transfection with SIRT5 small interfering RNA (siSIRT5) as compared to control siRNA (siSCR). (B) PAI-1 mRNA levels are induced in siSCR HAECS by treatment with 10 ng/mL TNF-α, this effect is lost upon SIRT5 silencing. (C) Accordingly, PAI-1 protein levels were increased in stimulated siSCR-treated cells while remaining similar to baseline in stimulated SIRT5-silenced HAECs. (D) Phosphorylation levels of the AMPK activator sub-unity α were higher in stimulated SIRT5-silenced HAECs than in control cells. (E–G) MAP kinases phosphorylation was induced by TNF-α in siSCR-treated control cells, in SIRT5-silenced HAECs this effect was specifically lost for ERK1/2 while not for p38 and JNK. n = 8–10 independent experiments. (A) unpaired two-tailed Student’s t-test; (B–G): one-way analysis of variance (ANOVA) with Bonferroni post hoc test. *P < 0.05, **P < 0.01. ERK1/2, extracellular signal-regulated kinases; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAECs, human aortic endothelial cells; JNK, c-Jun N-terminal kinases; MAP, mitogen-activated protein; PAI-1, plasminogen activator inhibitor 1; TNF-α, tumour necrosis factor α.