Figure 6. VASH1 controls SIRT1 level and increases stress resistance of HUVECs.

(A) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 72-hour incubation, Western blotting for VASH1 and SIRT1 was performed. (B) HUVECs were transfected with VASH1 siRNA or control siRNA. After a 24-hour incubation, SIRT1 activity was determined (*P<0.01, N = 3) (C) HUVECs were preteated with vehicle or 5 µmol/L SIRT1 activator 3 for 12 hours, and then transfected with VASH1 siRNA or control siRNA. After a 6-day incubation, SA β-gal staining was performed. Scale bars are 100 µm. SA β-gal-positive HUVECs were quantified, and the % of senescent cells was calculated (*P<0.01, N = 4). (D) HUVECs were transfected with SIRT1 siRNA or control siRNA. After a 72-hour incubation, Western blotting for VASH1 and SIRT1 was performed. (E) HUVECs were infected with AdVASH1 or AdLacZ. After a 72-hour incubation, Western blotting for VASH1 and SIRT1. (F) HUVECs were infected with AdVASH1. After a 24-hour incubation, HUVECs were then transfected with SIRT1 siRNA or control siRNA. After a subsequent 24-hour incubation, the cells were exposed to 100 µmol/L H₂O₂ for 1 hour followed by a 48-hour incubation in growth medium. Scale bars are 250 µm. SA β-gal staining and Western blotting for VASH1 and SIRT1 were then performed. β-gal-positive HUVECs were quantified, and the % of senescent cells was calculated (*P<0.01, N = 3). All the studies were repeated at least 3 times to confirm the reproducibility.