Research Paper Volume 16, Issue 2 pp 1352—1373

Figure 2. SIRT1 deacetylated Beclin-1 to enhance autophagy and injury of H/R-treated PMVECs. (A) Western blot showing the expression of SIRT1, Beclin-1, and Beclin-1-Ac under normoxia or H/R. (B) Western blot showing expression of Beclin-1 and Beclin-1-Ac under treatment with SRT2104 or Selisistat. (C) Western blot showing SIRT1 expression in PMVECs transfected with sh-SIRT1. (D) Detection of the interaction of SIRT1 with Beclin-1 in PMVECs with oe-SIRT1 under normoxia or H/R condition by Co-IP assay. (E) Beclin-1 protein stability analysis based on CHX treatment. (F) Western blot showing SIRT1, Beclin-1, and Beclin-1-Ac expression in H/R-treated PMVECs with oe-Beclin-1 and sh-SIRT1. (G) The mRFP-GFP-LC3 dual fluorescence system was used to trace autophagy formation after transfection of sh-SIRT1 or oe-Beclin-1 (magnification, 400 ×; scale bar = 25 μm). (H) Detection of changes in cell proliferation by CCK-8 assay after transfection of sh-SIRT1 or oe-Beclin-1. (I) Detection of PMVEC migration by Transwell assay (magnification, 200 ×; scale bar = 50 μm) after transfection of sh-SIRT1 or oe-Beclin-1. (J) Detection of apoptosis by flow cytometry after transfection of sh-SIRT1 or oe-Beclin-1. (K) Detection of Bax, Bcl-2, cleaved caspase-3 and caspase-3 expression by Western blot after transfection of sh-SIRT1 or oe-Beclin-1. Measurement data are expressed as mean ± standard deviation. Differences between 2 groups of data were compared using an unpaired t-test. Changes between multiple groups were compared using one-way ANOVA and Tukey’s multiple comparison test. Two-way ANOVA was used for data comparison at different time points. * p < 0.05. The cell experiments were repeated three times.