Figure 8.

Src and CDK3 mediate phosphorylation of TAZ on Ser89 under hypoxia in basal A cells. (A) Western blot analysis of p-TAZ (Ser89) in HCC1143 cells treated with the indicated SMARTpool siRNAs targeting components of the Hippo signaling cascade or luciferase or GAPDH as controls and exposed for 5 days to normoxia (NX) or hypoxia (HX) from two biological replicates. β-actin serves as loading control. (B,C) Western blot analysis of the indicated (phospho-)proteins in the indicated basal A (B) or luminal breast cancer cell lines (C) exposed to normoxia or hypoxia for 3 days in absence or presence of 1µM PP2. Tubulin serves as loading control. (D) Western blot analysis of the indicated (phospho-)proteins in HCC1143 basal A cells exposed to normoxia or hypoxia for 3 days in absence or presence of 50nM Dasatinib. (E) Top 10 kinases identified as candidate PP2 substrates by ChEMBL ranked by activity. * Indicates kinases where interaction with Dasatinib scored higher interaction with PP2. (F,G) Western blot analysis of TAZ and p-TAZ (Ser89) in HCC1143 cells treated with control (kinase pool) or CDK3 targeting SMARTpool siRNAs (F) or in HCC1143 cells transduced with control non-targeting or two different CDK3 targeting lentiviral shRNA constructs (G). Cells were exposed for 5 days to hypoxia. Graphs show qPCR analysis of gene silencing efficacy of the CDK3 targeting siRNA SMARTpool (F) or the CDK3 targeting lentiviral shRNA constructs (G). Mean expression relative to β-actin is shown. Error bars indicate SD for triplicate measurements. *, p < 0.05; **, p < 0.01.