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. 2015 Jun 22;5:542–549. doi: 10.1016/j.fob.2015.06.007

Fig. 1.

Fig. 1

Dasatinib, fluvastatin, and pazopanib inhibit the function of YAP/TAZ transcriptional co-activator. (A) Identification of small molecule agents which inhibit the nuclear localization of YAP. MDA-MB-231 cells were treated with a chemical library for 8 h. YAP was immunostained. Red: DNA, green: YAP. Each agent was used at 10 μM. (B) Dasatinib, fluvastatin, and pazopanib inhibited the nuclear localization of YAP and TAZ. MDA-MB-231 cells were treated with inhibitors for 8 h and YAP and TAZ were visualized by immunofluorescence. (i) Representative images of immunofluorescence. Bar represents 10 μm. (ii) Cells with nuclear localization of YAP and TAZ were counted. At least 75 cells were counted for each sample. Data represents mean and standard deviation from three independent experiments. (C) Inhibition of TEAD-dependent promoter activity by dasatinib, fluvastatin, and pazopanib. MDA-MB-231 cells were transfected with 8xGTIIC-luciferase TEAD reporter plasmid with pRL-CMV control plasmid, and treated with inhibitors overnight. Relative luciferase activity (8xGTIIC-luciferase/Renilla luciferase) was measured. Data represents mean and standard deviation from at least three independent experiments. *P < 0.02. (D) Inhibition of endogenous target genes of YAP and TAZ by dasatinib, fluvastatin, and pazopanib. MDA-MB-231 cells were treated with inhibitor for 18 h and total RNA was prepared. CTGF mRNA was quantified by quantitative RT-PCR. Data represents mean and standard deviation from at least three independent experiments. *P < 0.02. (E) Dasatinib, fluvastatin, and pazopanib activate the Hippo pathway. (i) Induction of phosphorylation of YAP and TAZ by dasatinib, fluvastatin and pazopanib. MDA-MB-231 cells were treated with inhibitors for 8 h. Whole cell extract was analyzed by SDS–polyacrylamide gel containing Phos-tag acrylamide. Asterisks show phosphorylated YAP and TAZ. Data is representative of at least three independent experiments. (ii) Degradation of YAP and TAZ by pazopanib is proteasome-dependent. MDA-MB-231 cells were treated with pazopanib for 8 h in the presence or absence of 10 μM MG132. Whole cell extract was analyzed by Western blot. Data is representative of at least three independent experiments.