2.

H₂O₂ and TSA modulate the expression of p53 and p21^WAF1 and the acetylation status of core histones in HCT116 p53⁺/⁺ cells.(A) H₂O₂ and TSA induce p53 and p21^WAF1 protein expression.(B–C) While H₂O₂ and TSA have no significant effect on p53 mRNA expression (real-time PCR, LightCycler), single H₂O₂ addition and TSA pre-treatment significantly increase p21^WAF1 mRNA expression. (D) Western Blotting showing that neither H₂O₂ nor TSA had a significant effect on the acetylation form of H3 and H4. (E) Western Blotting showing that H₂O₂ modulates the acetylation levels of p53 (Lys320, Lys382) and TSA reinforces this effect. The protein amounts of HDAC1 remain unchanged during all treatments.(F) Histone deacetylase (HDAC) activity of HCT116 p53⁺/⁺ cells was measured with a HDAC fluorimetric assay. The control cell level (C) was adjusted to 100%. *P<0.05. As expected TSA decreases HDAC activity.