Figure 3. Endogenous wild type p53 suppresses FoxM1 expression whereas R248Q mutant p53 enhances FoxM1 expression.

(A) Effects of p53 knockdown using shRNAs on FoxM1 protein expression in p53 WT A2780, NCI-H23, and p53 mutant HEC-1A and ES2 cell lines. Endogenous FoxM1 is upregulated when wild type TP53 is knocked down by two shRNAs in A2780 and NCI-H23 cells. Similarly, downregulation of S241F mutant TP53 also upregulate FoxM1 expression, suggesting that S241F mutant retains negative regulatory effect on FoxM1 expression. In contrast, downregulation of R248Q in HEC-1A cells downregulates FoxM1 expression, suggesting that R248Q mutant may have gain of positive regulatory effect of FoxM1 expression. Proteins were isolated from p53 shRNA batch clones and subjected to Western analysis. β-actin was used for normalization of loading. (B) p53 knockdown using shRNAs blocks the downregulation of FoxM1 protein expression by Nutlin-3 in A2780 and NCI-H23 cells. Cells were treated with or without Nultin-3 for 24 h. Proteins were then isolated and subjected to Western analysis. β-actin was used for normalization of loading. (C-D) p53 knockdown blocks the downregulation of FoxM1 mRNA by Nutlin-3 in A2780 and NCI-H23 cells. Cells were treated with or without Nultin-3 for 24 h. Total RNA was isolated and subjected to real-time RT-PCR analysis. GAPDH was used for normalization of FoxM1 expression. Data are presented as Mean ± SD of 3 experiments. * indicates P < 0.05 by paired t-test.