Figure 2. FoxM1 mRNA is downregulated by Nutlin-3 in TP53 wild type cells but not in TP53 mutant cells.

Upper panel (A-C): Cells were treated with vehicle (0.05% DMSO) or 10 μM Nutlin-3 for 3, 6 or 24 h. Lower panel (D-F): Cells were treated with DMSO, Nutlin-3, CHX, CHX+Nutlin-3, ActD or ActD+Nutlin-3 for 24 h. Total RNA was isolated and subjected to real-time RT-PCR analysis. GAPDH was used for normalization of FoxM1 expression. It is important to note that FoxM1 mRNA is quite stable in the presence of ActD in cell lines with wild type TP53 (D&E). Data are presented as Mean ± SD of 3 experiments. **** indicates P < 0.0001 by paired t-test. Difference alphabet letters indicate significant differences (P < 0.05) across treatments by One-Way ANOVA. Nutlin-3 treatment enhances FoxM1 mRNA decay. (G) Analysis of the effect of Nutlin-3 on FoxM1 mRNA stability using actinomycin D. A2780 cells were treated with or without Nutlin-3 for 14 hours, followed by actinomycin D treatment for 30, 60, 120, or 240 min. Total RNA was isolated and subjected to real-time RT-PCR analysis of FoxM1. cMyc mRNA was used as a positive control. (H) Effects of Nutlin-3 on nascent FoxM1 transcription in A2780 cells. Click-iT EU labeling followed by real-time RT-PCR was used to compare nascent FoxM1 mRNA synthesis at 2 h or 5 h after treatment with vehicle or Nutlin-3. * indicates P < 0.05; ** indicates P < 0.01. (I) Effects of Nutlin-3 on FoxM1 mRNA decay. A2780 cells were treated with or without Nutlin-3 for 5 h before pulse-labeling for 1 h. Total RNA was isolated at 0, 2, 8, or 20 h post labeling and subjected to RT-PCR analysis. FoxM1 mRNA half-life was calculated using GraphPad Prism6.