Figure 3.

MDM2 inhibitors increase expression of downstream target genes of p53 in hypoxic p53^WT cancer cells. Gene expression was measured by qPCR in (a, b) HCT116 p53^+/+ and p53^−/− cells, and in (c, d) B16-F10 p53^+/+ and p53^−/− cells after 24 h treatment with MDM2 inhibitors. Human cells (HCT116) were treated with 2 µM nutlin-3a or 0.5 µM navtemadlin, while mouse cells (B16-F10) were treated with 10 µM nutlin-3a or 2 µM navtemadlin. Each data point represents averaged values of duplicates from three to five independent experiments. Expression was normalized to house-keeping genes B2M or Rplp0 for human or mouse cell lines, respectively. Statistical significance was assessed on non-log transformed dCT values using 2-way ANOVA followed by post-hoc Dunnett's correction for multiple testing (unpaired, two-tailed, α = 0.05). Adjusted P values for individual comparisons (e.g., DMSO vs nutlin-3a) from the Dunnett’s test are indicated (*P < 0.05; **P < 0.01; ***P < 0.001). For Cdkn1a in c, d, values indicate the source of variation identified by the ANOVA (run prior to the post-hoc t test); post-hoc comparisons of each drug against DMSO did not provide significant values, likely due to insufficient power.