Fig. 3: kbhb reduces the acetylation of p53.

a H1299 cells were transfected with Flag-p53 and then treated with 10 mM BHB for 4 h, with 10 µM Ex527 (an inhibitor of SIRT1), for 2 or 4 h, or with both BHB and Ex527. p53 was purified and enriched by IP. Western blot analysis using BHB-K and Ac-K antibodies of purified p53 (M2 IP) or whole cell lysates (lysate) with different exposure time (s: shorter exposure time; l: longer exposure time). b Quantification of acetylation p53 protein levels to normalize to p53 protein levels by western blot represented in (a). Data shown are averages of three times + SEM. The p value was determined by paired Student’s t test (**p < 0.005, ***p < 0.001). c HCT116 cells were treated with 10 mM BHB for 4 h, 10 µM Ex527 for 2 or 4 h, or with both BHB and Ex527. Cell lysates were IP with BHB-K or Ac-K antibodies. Western blot using anti-p53 DO-1 antibodies of Ac-K and BHB-K IP, or of the whole cell lysate with different exposure time (s: shorter exposure time; l: longer exposure time). d Quantification of acetylation p53 protein levels to normalize to p53 protein levels by western blot represented in (c). Data shown are averages of three times + SEM. The p value was determined by paired Student’s t test (***p < 0.001). e Western blot analysis to determine the level of acetylated p53 using Ac-p53 antibodies, anti-p53 in p53-purified IP obtained from cells transfected with p53 or both p53 and CBP and then treated with 10 mM BHB for the indicated time. IP were performed with M2 beads. Western blot of whole cell lysates using anti-p53, anti-HA (to detect CBP) and anti-actin is also shown (bottom). f HCT116 cells were treated with 10 mM BHB for 4 h, 10 µM Ex527 for 2 or 4 h, or both BHB and Ex527. The expression levels of p53, actin (as a loading control) and the p53 downstream genes p21 and PUMA were analyzed by western blot. g The levels of the p53 downstream genes p21 and PUMA were decreased in the p53/H1299-inducible cell lines regardless of whether they were untreated, treated with BHB, treated with doxycycline to induce p53 expression, or treated with BHB and doxycycline together. h p21 and PUMA expression were decreased in HCT116 cells treated with BHB, Doxorubicin to damage DNA to activate p53, or both BHB and Doxorubicin. i BHB treatment decreased the p21 and PUMA expression induced by nutlin in U2OS cells