Figure 3.

HPV16E7 promotes phosphorylation of YAP. (A) Western blot analysis of the expression level of YAP and p-YAP in Mock-transfected cells, and in cells transfected with E7wt or deletion mutant constructs E7Δ62–66 or E7Δ71–75. β−actin determination was used as loading controls. Bar graph (bottom) shows relative densitometry quantitation of each protein normalized to β-actin obtained in three independent experiments. * p < 0.05 and ** p < 0.01, vs. Mock transfected cells. (B) Bar graph showing flow cytometry evaluation of YAP and p-YAP amount restricted to pAmCyan-positive cells. Results are the means ± SD of the median fluorescence intensity obtained in four different experiments. ** p < 0.01, and *** p < 0.001, vs. Mock transfected cells. (C) IVM analysis after double cell staining with anti-YAP (upper line) or anti-p-YAP antibody (bottom line) (both in red) and Hoechst (blue) of cells transfected (green) with pAmCyan empty vector (Mock), E7wt or deletion mutant constructs E7Δ62–66, E7Δ71–75. Interestingly, p-YAP is found exclusively in the cytoplasm only in E7wt cells (yellow fluorescence, see arrow). (D) Bar graphs showing the evaluation of mRNA levels of Cyr61 (left) and CTGF (right), two downstream YAP target genes, performed by real-time qRT-PCR assay. Data are reported as mean ± SD of RNAs relative fold change vs Mock-transfected cells, obtained in three independent experiments. ** p < 0.01 and *** p < 0.001 vs. Mock transfected cells. Uncropped western blot images for Figure 3A available in Supplementary Figure S7.