Figure 2. MiR-542-3p increases the stability of p53 protein.

(A) MiR-542-3p induces p53 at protein level. U2OS cells were transfected with negative or miR-542-3p mimics at indicated concentration. Cells were pelleted for western blotting of p53 and p21. (B) MiR-542-3p increases the stability of p53 protein. U2OS cells transfected with either negative or miR-542-3p mimics at 10 nM were reseeded and treated with protein synthesis inhibitor cycloheximide (CHX, 20μg/ml) to block de novo protein synthesis. Cells were harvested at indicated time for western blot analysis. Half-life of p53 was determined by densitometric analysis of p53 bands from three independent experiments using the formula t_1/2=−Ln2/S, where S represents the slope from each linear regression. (C) MiR-542-3p failed to further increase p53 level in the presence of MG132. U2OS cells transfected with either negative or miR-542-3p mimics at 10 nM were reseeded and treated with MG132 (2μM) before western blot analysis of p53. (D) MiR-542-3p reduced MDM2-p53 interaction and p53 poly-ubiquitination. HCT116 cells transfected with either negative or miR-542-3p mimics were treated with MG132 (2μM) for 4hr. Cells were then lysed for immunoprecipitation with anti-p53. Immunoprecipitated proteins were subjected to western blot analysis of MDM2 and polyubiquitinated p53 ((Ub)n-p53). Quantitation of protein levels was labeled below the corresponding blots (same for Fig.3–5).