Figure 8.

p53 SUMOylation at K386 mediates AOPP-BSA-induced endothelial senescence. (A) HUVECs were infected with adenovirus of K386R, WT-TP53, or Ad-GFP for 48 h. The effects of K386R, WT-TP53, and Ad-GFP on the levels of p53 and flag were detected by western blot. GAPDH was used as loading control. (B,C) Quantification of p53 and flag protein expression (n = 4). (D,E) At 48 h post-infection, HUVECs were incubated with or without 200 μg/ml AOPP-BSA for 12 h. The protein levels of p53 and p53 SUMOylation were detected by western blot and immunoprecipitation. (F) Quantification of p53 protein expression (n = 4). (G) The SUMO1-p53/p53 ratio was calculated (n = 3). (H) HUVECs were infected with K386R, WT-TP53, and Ad-GFP with or without AOPP-BSA stimulation. The expression of senescence-associated proteins p21 and p16 were detected by western blot. (I,J) Quantification of p21 and p16 protein expression (n = 4). (K) HUVECs were treated as mentioned above, and the SA-β-gal assay was used to identify the senescent cells. Scale bar for upper panel = 20 μm, scale bar for lower panel = 50 μm. (L) The SA-β-gal positive rate was quantitated (n = 4). (M) HUVECs were incubated with or without 200 μg/ml AOPP-BSA for 12 h, then the expression of p53 and acetylation of p53 were detected by western blot. (N) The ratio of acetylated p53 and total p53 was calculated (n =6 ). Two-tailed unpaired Student's t-test for (N). One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons for (B,C,E,G,I,J,L). Data are shown as mean ± SEM. ns, non-significant (P > 0.05). Ad-GFP, control adenovirus containing GFP; WT-TP53, wide-type TP53 adenovirus; K386R, TP53 K386-deficient adenovirus; Ac-p53 (K386), acetylated p53 at K386.