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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting CSCs compartment via YAP inhibition

Fig. 3

Valproic acid/simvastatin combination impairs YAP activity via modulation of mevalonate pathway and of AMPK. a The network was generated by Ingenuity Pathway Analysis (IPA) using “mevalonate pathway enzymes” and “HDAC inhibitors” search. Network genes are visualized by proper symbols, which specify the functional nature of the correspondent protein. Each node represents a gene and its direct (represented by solid lines) and indirect (represented by dotted lines) association with other genes. b Western blot analysis of p53, phospho AMPK (pAMPK), AMPK, phospho ACC (pACC), ACC, phosphoHMGCR (pHMGCR), phospho YAP (pYAP) and YAP in 22Rv1 treated with VPA and/or SIM at the IC5096h doses for 4 h. Extracts (30 μg) were resolved by SDS-PAGE and immunoblotted using specific antibodies, βactin or γtubulin were used as loading control. Densitometric analysis was performed by ImageJ image software and reported as ratio relative to the indicated loading control. c CTGF, CYR61, BIRC5 and NANog mRNA expression was evaluated by RT-PCR after 4 h of cell culture in absence or presence of VPA and SIM at IC5096h doses. β-actin was used as housekeeping control gene to normalize RT-PCR reactions. d Expression of phospho-YAP (pYAP) and YAP evaluated by western blotting in 22Rv1 1st generation spheres untreated or treated for 4 h with VPA, SIM or the combination at the IC5096h doses. Γtubulin was used as loading control. Densitometric analysis was performed by ImageJ image software. e BIRC5 mRNA expression evaluated by RT-PCR in 22Rv1 1st generation spheres untreated or treated for 4 h with VPA, SIM or their combination at the IC5096h doses. β-actin was used as housekeeping control gene to normalize RT-PCR reactions. f Surface marker expression (CD44 and CD113) was determined by flow cytometry on 22Rv1 1st generation spheres untreated or treated for 4 h with VPA, SIM or their combination at the IC5096h doses. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

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