Fig. 4
Constitutively active YAP5SA mutant reverts the effect of valproic acid and simvastatin combination. a Western blot analysis of phopho-YAP (pYAP), and YAP basal levels in control, wild-type YAP (wt-YAP)-transfected and YAP5SA (constitutively active)-transfected 22Rv1 cells. GAPDH serves as control for equal protein loading. Densitometric analysis was performed by ImageJ image software. b Western blot analysis of pYAP, YAP, Caspase 3 and PARP in 22Rv1 control, wt-YAP and YAP5SA cells, untreated or treated for 4 h with VPA and/or SIM at the IC5096h doses. Βactin serves as control for equal protein loading. c YAP-immunofluorescence microscopy detection in 22Rv1 control, wt-YAP and YAP5SA cells untreated or treated indicated above. Cells were stained with anti-YAP antibody (green-Alexafluor488) and DAPI for nuclei detection (blue) and detected as indicated in Methods section. d CTGF and CYR61 mRNA expression evaluated by RT-PCR after 4 h of cell culture in absence or presence of VPA and SIM at the IC5096h doses. The values are means ± S.D. of technical triplicates and Dunn’s multiple comparisons test was performed to compare the three groups for each genes. e Limiting dilution assay performed on 22Rv1 control and 22Rv1-YAP5SA cells, untreated or treated for 24 h with VPA/SIM at the IC5096h doses and plated in ultra-low 96-well without additional treatment for three weeks. Clonal frequency was evaluated with the Extreme Limiting Dilution Analysis ‘limdil’ function as described in Methods section. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)