Figure 5

Effect of overexpression of YAP on BMP-2 signalling. C3H10T1/2 cells were transduced with retrovirus encoding hYAP1, hYAP1(S127A), hYAP2 or hYAP2(S127A), and GFP separated from the YAP gene by an IRES sequence. Cells transduced with empty vector (EV) retrovirus and/or cells left non-transduced (NT) served as controls. Cells were plated in micromass culture and the next day treated with 300 ng/ml BMP-2 or vehicle only. (A) pSmad1,5,8 and total Smad1 after 1 h of BMP2 treatment determined by western blotting. (B, C) Ratio of pSmad1,5,8/Smad1 after 1 h of BMP2 treatment shown as mean ± standard deviation (SD) (n = 3). ^** P <0.01; ^*** P <0.001. (D) Time-course of BMP-2-induced pSmad1,5,8 and total Smad1. (E) Expression of the BMP target genes Inhibitor of differentiation (Id)1, Id2 and Id3 after 4 h of BMP-2 treatment determined by quantitative RT-PCR. Data was normalised to ACTB expression, and is shown as mean ± SD (n = 4). ^*** P <0.001. BMP, bone morphogenetic protein; GFP, green fluorescent protein; pSmad, phosphorylated Smad; pYAP, phosphorylated YAP; RT-PCR, reverse transcription PCR; YAP, Yes-associated protein.