Figure 4. miR-34a overexpression increases acetylation of p53 and p53-DNA binding activity.

Mouse NS cells at 3 days of differentiation were transfected with either SIRT1 siRNA or scrambled control for 48 h or with pre-miR-control or pre-miR-34a for 72 h. Cells were then collected for total and nuclear protein extraction or stained with Annexin-V-APC/PI to evaluate cell death. p53-overexpressing cells were used for supershift and competition experiments. Radio-labeled double-stranded oligonucleotide corresponding to the p53 consensus (p53 -cons) was used as a probe. A. Representative immunoblot showing increased p53 acetylation at lysine 379 after SIRT1 silencing or overexpression of miR-34a. Ponceau S was used as loading control. B. Representative EMSA showing the specificity of the complex formed with p53-cons probe. Supershift experiments were performed using an anti-p53 antibody (DO-1, Santa Cruz Biotechnology). Competition experiments were performed by adding 10- or 100-fold excess of unlabeled double-stranded oligonucleotides bearing a mutation in the consensus site (p53-cons-mut), or containing either two or one quarter-sites known to be consensus sites for p53 (p53-A and p53-B, respectively) or a non-specific sequence (NS). C. EMSA showing increased p53-DNA binding activity in pre-miR-34a transfected cells for 48 and 72 h. D. Representative Annexin V-APC/PI staining showing absence of cell death after miR-34a modulation.