Figure 3.

Increase of the p53 response to DNA replication inhibition following ASPP1 and YAP down-regulation. (A) Cell cycle of HCT116 treated for 24 h with 400 μM of HU or 10 μM of Nutlin, analysed by BrdU and PI incorporation and measured by flow cytometry. Result shows a typical histogram for the different conditions. (B) HCT116 cells transfected by control siRNA or siRNA against ASPP1 and treated as in (A) were analysed by western blot with specific antibodies against ASPP1, p53 and p53 acetylated on residues K373/K382. (C) HCT116 cells were treated as in (B) and mRNA expression of p21 was tested by RT–qPCR using specific primers. The results were normalized against two different standard genes, and the graphs represent the mean of three independent experiments. (D) p21 protein expression was measured by western blot. Actin was used as a loading control. (E) Cell cycle of HCT116 transfected with control or ASPP1-directed siRNA and treated 24 h with HU, analysed by BrdU and PI incorporation measured by flow cytometry. Results represent the mean of three independent experiments. (F) Cells were treated as in (E), then cells with an S-phase DNA content were gated and analysed for BrdU incorporation by flow cytometry.