Fig. 3. p63 overexpression cannot rescue E7-induced cell cycle arrest.

a CaSki cells were transiently transfected with si-E7 and cell proliferation was assessed at indicated time points. The graph shows the average number of cells in triplicate 24 wells as compared to scrambled-transfected controls. b Cell death following E7-depletion was assessed by flow cytometry as described in section “Materials and methods” and also by western blot analysis for cleaved PARP. c E7-depletion results in a significant increase in the number of cells undergoing G1-arrest as detected by flow cytometry. Cells were collected at indicated time points following transfection of CaSki cells with siE7 or scrambled control siRNA (scr). Graph shows percentage of cells in G1 following E7-depletion at indicated time points (***P-value < 0.001, ****P-value < 0.0001 calculated by Student’s t-test). d CaSki cells were transfected as indicated and collected after 5 days. N-TERT cells were induced to differentiate by adding 1.2 mm Ca²⁺ and were harvested after 7 days. Cell lysates from scr/siE7-transfected cells and proliferating and differentiated N-TERT cells were analysed for the expression of the differentiation marker involucrin (inv). e G1 arrest was induced in CaSki cells by treating with 6 μM aphidicolin (APH) or by contact inhibition (CI) (growing cells at confluency for up to 7 days). p63 expression was assessed by western blotting. f CaSki cells were transfected with siE7 and scrambled control siRNA in duplicates. Twenty-four hours later one plate of cells from each duplicate was re-transfected with pcDNA3.1 empty vector and the other plate of cells with HAp63-pcDNA3.1 plasmid. Cells were collected 48 h later and expression of E7 and p63 was revealed by western blotting. g A fraction of cells was used to assess cell cycle profile by flow cytometry. Amount of cells in G1 was quantified and plotted on a graph to demonstrate impact of p63 overexpression on cell cycle. h Proliferation assay showing the effect of p63 over-expression in E7-depleted cells compared to scrambled siRNA and/or pcDNA3.1 empty vector transfected control cells. (Error bars represent s.e.m. in all graphs.)