a HBMVECs were transfected with scrambled siRNA or Sirt1 siRNA for 24 h, and the levels of Sirt1 and HMGB1 in cell lysates were analysed by western blotting; β-actin was used as a loading control. b HBMVECs were transfected with empty vector (E.V.), wild-type SIRT1 (WT Sirt1) or dominant negative mutant SIRT1 (DN Sirt1) plasmids, as described in the methodology. The levels of Sirt1 and HMGB1 were analysed by western blotting in the genetically modified HBMVECs with CHX treatment (100 μg/ml) for the indicated times. Representative blots are presented, and graph indicate the relative levels of HMGB1 at each time point as compared to 0 hr of each group (n = 3–4, mean ± SD; **p < 0.01 among groups by two-way ANOVA). c–f Using HBMVECs transfected with WT SIRT1 for 24 h, cells were harvested and then analysed by western blotting using an anti-LC3 antibody (c n = 3, mean ± SD; *p < 0.05 versus E.V. by unpaired t-test). HBMVECs with WT SIRT1 transfection were treated with lysosome inhibitors (d n = 3-4, mean ± SD; *p < 0.05 versus WT Sirt1 without lysosomal inhibitors by unpaired t-test), bafilomycin A1 (Baf.A, e n = 3, mean ± SD; *p < 0.05 versus WT Sirt1 without Baf.A by unpaired t-test; #p < 0.05, ##p < 0.01 versus without treatment by one-sample t-test), or MG132 (f n = 3, mean ± SD), and the expression levels HMGB1 were analysed. In Baf.A-treated cells, the levels of p62, and LC3 were further analysed by western blotting (e). β-Actin was used as a loading control. All blots are representatives from repeated experiments.