Fig. 4From: Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting CSCs compartment via YAP inhibitionConstitutively 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)Back to article page