Figure 5.

Exogenous expression of SAV1 inhibits YAP transcriptional activity by stabilizing MST1 protein. (A) WB was used to analyze the effect of SAV1 overexpression on Hippo signaling pathway components in H2170 and HCC827 cells. (B) The effect of SAV1 overexpression on the subcellular localization of YAP was determined by immunofluorescence (IF) staining for endogenous YAP (red) along with DAPI for DNA (blue), Scale bar: 50 µm. (C) The effect of SAV1 overexpression on the subcellular localization of YAP was determined by WB. (D) Luciferase reporter assay (8XGTIIC) in NSCLC cell lines transfected with SAV1 and without. (E, F) Lung cancer cell lines with overexpression and none-overexpression SAV1 were treated with cycloheximide (CHX, 200 µM) for 0, 2, 4, and 6 hours, and lysates were subjected to western blot as indicated. GAPDH was used as the loading control. (Right) The relative quantitation of MST1 after CHX treatment. (G) Immunoprecipitation of MST1 protein in overexpression and none-overexpression SAV1 cell lines and determination of its ubiquitination by immunoblotting with anti-ubiquitin in the presence or absence of the proteasome inhibitor MG132 (50 µM). Results represent means ± SD of at least two independent experiments. Statistical significance: p< 0.05 (Student's t-test).