Figure 3

Effect of overexpression of YAP on chondrogenic differentiation of C3H10T1/2 MSCs. C3H10T1/2 cells were transduced with retrovirus encoding hYAP1 or hYAP1(S127A) and GFP separated from the hYAP1 gene by an IRES sequence. Cells transduced with empty vector (EV) retrovirus and/or cells left non-transduced (NT) served as controls. (A) Detection of YAP, pYAP, and GFP by western blotting. (B-D) Cells were treated with 300 ng/ml BMP-2 (B, D) or 10 ng/ml TGF-β1 (C) in micromass culture for 7 days to induce chondrogenic differentiation. (B, C) Whole-mount alcian blue staining followed by extraction and measurement of absorbance at 630 nm. Absorbance was normalised to DNA content determined from parallel micromass cultures. Data is shown as mean ± standard deviation (SD) (n = 4), and expressed relative to the NT (B) or EV controls (C). (D) Chondrogenic marker expression by quantitative RT-PCR. Data is shown as mean ± SD (n = 3). ^** P <0.01; ^*** P <0.001. BMP, bone morphogenetic protein; GFP, green fluorescent protein; MSC, mesenchymal stromal/stem cell; pYAP, phosphorylated YAP; RT-PCR, reverse transcription PCR; TGF, transforming growth factor; YAP, Yes-associated protein.