Figure 6
New synthesized compounds induce p53 protein accumulation by dissociation of MDM2/p53 complex and stimulate transcription of p53 target genes in U87MG cells.
The U87MG cells were treated with DMSO (control sample) or the indicated concentrations of the compound 1 or 7 for 12 h. Lysates were subjected to Western blot analysis using antibody to p53 (FL-393; Santa Cruz Biotechnology). One representative Western blot is presented (panel (A) for each cell treatment. β-actin was used as the loading control. The bar graph (panel (B) shows the quantitative analysis of the Western blots, performed using ImageJ. Data are expressed as the percentage of OD versus control set to 100% and represent the mean ± SEM of three different experiments. Statistical significance was determined with a one-way ANOVA with Bonferroni post-test: *P < 0.05, ***P < 0.001 vs Control; (C) Evaluation of MDM2/p53 complex: U87MG cells were incubated with compound 1 (5 μM) for 8 h followed by immunoprecipitation using an anti-MDM2 antibody. The MDM2/p53 complex and the relative input of the proteins were detected by immunoblot. One representative Western blot is presented (left panel). The bar graph (right panel) shows the quantitative analysis of the Western blot, performed using ImageJ. Data represent the mean ± SEM of three different experiments. **P < 0.01 vs Control. Full-length blots are reported in Supplementary Information section titled “Full-length blots relative to the cropped images showed in the main Figures”. (D) Relative mRNA quantification of p53 target genes: The relative mRNA quantification of p53 target genes (p21 and MDM2) was performed by real-time RT-PCR as describe in Methods. Data represent the mean ± SEM of three different experiments. Each experiment was performed in duplicate. Statistical significance was determined with a one-way ANOVA with Bonferroni post-test: *P < 0.05, **P < 0.01 vs Control.
