Research Paper Volume 15, Issue 20 pp 11184—11200

Figure 5. Effect of SIRT1 deacetylation on p65 in H₂O₂-induced pyroptosis of HAECs. (A) Western blot demonstrating the increased protein expression ratio of Ac-p65 (Lys310)/p65 in the H₂O₂ group compared with the NC group. The overexpression of SIRT1 protein levels decreased the protein expression ratio of Ac-p65 (Lys310)/p65 compared with that in cells treated with H₂O₂. (B) Endogenous IP Western revealed the pulling down of the HAEC extracts with either a negative control (IgG) or RelA/p65 antibody, followed by SIRT1 or RelA/p65 immunoblot. (C) Western blot demonstrating the increased p65 protein levels in the cytoplasm of H₂O₂-treated HAECs compared with NC-treated cells. Overexpression of SIRT1 protein levels increased the p65 protein levels in the cytoplasm compared with that in cells treated with H₂O₂. (D) Western blot demonstrating increased p65 protein levels in the nucleus of H₂O₂-treated HAECs compared with NC-treated cells. Overexpression of SIRT1 protein levels decreased p65 protein levels in the nucleus compared with that in cells treated with H₂O₂. (E) Double staining of p65 (green) and DAPI (blue). DAPI staining results of the nucleus of HAECs. P65 staining results of increased p65 in the nucleus of H₂O₂-treated HAECs; the level decreased after transfection with ov-SIRT1 in H₂O₂-treated HAECs under a fluorescence microscope. Plotting scale =50 μm. (^*p<0.05 vs NC, ^**p<0.01 vs NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; NC, negative control; IP, immunoprecipitation; DAPI, 4′,6-diamidino-2-phenylindole.