Figure 2
p53 post-transcriptional silencing abrogates the G1 arrest after DNMT1 depletion. A) Real time RT-PCR and (B, C) Western Blot analysis revealed a strong increase of p21waf1-Cip1 and p53 protein levels in IMR90 cells and not in HCT116 cells at 72 hours siDNMT1 post-transfection. At 144 hours p53 level was reduced. Cells treated with adriamicyn to induce DNA damage were used to assess the functionality of the p53 pathway. D) Real time RT-PCR and detection of p53 and DNMT1 protein levels by WB (E) in siDNMT1/p53 (72 h) and control IMR90 cells. Summary graphs illustrating differences in p21waf1-Cip1 and p53 protein levels (see Additional files 2, 3, and 4 for the relative densitometric analyses). Data are expressed related to control cells following normalization with β-tubulin. F) Top panels: IMR90-ctrl and IMR90-siDNMT1/p53 cells pictures at 72 hours post-transfection showing that cell density/dish in IMR90-siDNMT1/p53 cells is comparable to that of IMR90-ctrl cells. Central panels: cytofluorimetric profiles showing no significative differences in cell cycle distribution of control and IMR90-siDNMT1/p53 cells. Bottom panel: histogram showing IMR90 siDNMT1/p53 cells proliferation in comparison to control and sip53 cells.