Research Paper Volume 15, Issue 20 pp 11184—11200

Figure 3. Effect of p65 on the H₂O₂-induced pyroptosis of HAECs. (A) Western blot demonstrating the increased protein levels of p65 upon H₂O₂ treatment in a time-dependent manner. (B) qRT-PCR results demonstrating the decreased mRNA expression of p65 in cells transduced with si-p65 compared with that in untransduced cells. (C) Western blot demonstrating the decreased protein expression of p65 in cells transduced with si-p65 compared with that in untransduced cells. (D) LDH release from HAECs treated with or without si-p65 following H₂O₂ treatment. (E) PI staining results of the increased percentage of PI positive cells in H₂O₂-treated HAECs. Double staining of PI (red) and Hoechst 33342 (blue). The percentage of PI positive cells in H₂O₂-treated HAECs decreased after transfection with si-p65. Plotting scale = 200 μm. (F) qRT-PCR results demonstrating the increased mRNA expression of NLRP3 and IL-1β in HAECs under H₂O₂ treatment, and decreased mRNA expression of NLRP3 and IL-1β in HAECs transduced with si-p65 compared with cells transduced with NC under H₂O₂ treatment. (G) Western blot results demonstrating the decreased protein expression of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 in HAECs transduced with si-p65 compared with cells transduced with NC under H₂O₂ treatment. Western blot demonstrating the increased protein expression of p65 in the cytoplasm (H) and nucleus (I) of H₂O₂-treated HAECs compared with NC-treated cells. The p65 protein levels in nucleus were obviously increased. (^*p<0.05 vs control or NC, ^**p<0.01 vs control or NC, ^***p<0.001 vs NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂. ^###p<0.001 vs H₂O₂). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; LDH, lactate dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction; PI, propidium iodide; NC, negative control.