Figure 3.

Src kinase is the most dominant therapeutic target of dasatinib that regulates YAP activity in RCC cells. (A) Caki-1 and 769-P cells were treated with a customized pre-designed genOFF™ RNAi library for 72 h. Cell viability was assessed by SRB assay. The mean viability of cells receiving different siRNAs for the same gene was evaluated by Log2 calculation. (B) Caki-1 and 769-P cells were transfected with scramble or Src siRNAs. Cell viability was measured using SRB assay. Bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (C) RCC cells were treated with dasatinib at 1 μM for 3 h followed by immunoblotting analysis. (D-E) Caki-1 and 769-P cells were stably transfected with retrovirus expressing either empty vector, Src-WT or Src-T341I. The resultant cells were treated with dasatinib (0.1 and 1 μM) for 3 h and the phosphorylation of Src was measured by immunoblotting (D). Cell viability after dasatinib treatment for 72 h was assessed by SRB assay. Bars, mean ± SD. ***P < 0.001 (E). (F-G) Caki-1 cells were treated with scramble or Src siRNAs for 72 h and then subjected to immunoblotting with the indicated antibodies (F). The subcellular location of YAP was determined by immunofluorescence. Scale bar, 50 μm (G). (H) Caki-1 cells stably expressing either empty vector, Src-WT or Src-T341I were treated with dasatinib for 3 h, and YAP phosphorylation was measured by immunoblotting.