Fig. 6

Overexpression of YAP1^S127A antagonizes the synergistic inhibitory effect of Roblitinib and CPI-169 in HCC cells. A Western blot analysis of FLAG-YAP1^S127A expression in HepG2 and SMMC-7721 cell lines stably transfected with empty vector control (Con) or FLAG-YAP1^S127A (YAP1^S127A). B qPCR analysis of YAP1 mRNA level in HepG2 and HepG2 YAP1^S127A cell lines following the CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ****p < 0.0001, ns, no significance). C Cell viability of HepG2, HepG2 YAP1^S127A, SMMC-7721 and SMMC-7721 YAP1^S127A cell lines was evaluated by the CCK-8 following increasing concentrations of CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3). D Drug interaction analysis between CPI-169 and Roblitinib in HepG2, HepG2 YAP1^S127A, SMMC-7721 and SMMC-7721 YAP1^S127A cell lines. The CI values less than 1.0, approximately 1.0 and greater than 1.0 indicate synergism, additive and antagonism, respectively. E EdU assays of HepG2 and HepG2 YAP1^S127A cell lines following the CPI-169 + Roblitinib treatment for 48 h. Scale bars: 50 ?m. F Colony formation assay of HepG2 and HepG2 YAP1^S127A cell lines following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 14 days. Scale bars: 1 cm. G Measurement of the cell numbers in (E). Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ***p < 0.001, ****p < 0.0001). H Measurement of the clone numbers in (F) and SMMC-7721 and SMMC-7721 YAP1^S127A cell lines following CPI-169 and/or Roblitinib treatment for 14 days. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, no significance). I-J Flow cytometric analysis of apoptosis in HepG2 and HepG2 YAP1^S127A cell lines following CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, **p < 0.01, ****p < 0.0001). K-L Zebrafish harboring KRAS^G12V+ (left) or KRAS^G12V+/YAP1^S87A+ (right) HCC primary tumors were treated with CPI-169 + Roblitinib for 72 h. Scale bars: 100 ?m. Data are presented as mean ± SEM (n = 12, two-way ANOVA with Sidak?s multiple comparison test, *p < 0.05, ****p < 0.0001 ns, no significance)