Figure 4

Downregulation of SAV1 activates YAP1. a Phosphorylation of YAP1 was decreased in RCC cell lines. Phosphorylation of YAP1 in HK2, 786-O and 769-P cells was analyzed by Western blot analysis with anti-phospho-YAP antibody, anti-YAP antibody, and anti-GAPDH antibody, respectively. Protein level of SAV1 in each cell line was demonstrated with anti-SAV1 antibody. SAV1 was detectable only in HK2 cells. b Phosphorylated YAP1 is increased in SAV1-transduced 786-O cells. 786-O cells were transduced with pLenti6.3/V5-DEST empty vector or SAV1-pLenti6.3/V5-DEST, and one clone of control cells (designated as the control) and two independent clones of SAV1-re-expressing cells (designated as SAV1-1 and SAV1-2) were established. Western blotting was performed with anti-phospho-YAP antibody, anti-YAP antibody, anti-SAV1 antibody and anti-GAPDH antibody, respectively. c SAV1 suppresses transcriptional activity of YAP1-TEAD3. SAV1-1, SAV1-2 and control cells were transfected with the Gal4-TEAD3 plasmid vector that expresses TEAD3 fused to GAL4 (kindly provided by Dr. B. Zhao [19]), Gal4-9x UAS luciferase reporter (pGL4.31), and pRL-CMV. Firefly luciferase activity was normalized to Renilla luciferase activity. Normalized luciferase activity in control cells untransfected with TEAD3 was set at 1. In SAV1-1 and SAV1-2 cells, the luciferase activity was significantly decreased. Experiments were performed in triplicate. The dual luciferase data are shown as mean ± SD. *p < 0.005 *; Student's t test.