Fig. 5
β-Catenin falling off in YAP-KO MC3T3 cells. a–d Western blot analysis of YAP and β-catenin (C7207, Sigma) in ctrl and Yap-KO MC3T3 cell lines. Yap-KO MC3T3 cell line is generated by CRISPR-Cas9 strategy. Total lysates (a, b) and lysates of nuclei and cytoplasmic fractions (c, d) of ctrl and Yap-KO MC3T3 cells were subjected to immunoblot analysis using the indicated antibodies. a, c, representative blots; b, d, quantification analysis (mean ± SD, n = 3-different cultures, *P < 0.05). e, f Co-immunostaining analysis of β-catenin (C2206, Sigma) and YAP in ctrl and Yap-KO MC3T3 cells. e, representative images. Scale bar, 10 µm. f, quantification data (mean ± SD, n = 50 from 3 different cultures). ***P < 0.000 1. g, h Reduced cell proliferation in Yap-KO MC3T3 cells revealed by immunostaining analysis of Ki67 (a marker for cell proliferation). g, representative images, Scale bar, 20 µm. h, quantification data (mean ± SD, n = 50 from 3 different cultures). ***P < 0.000 1. i Real-time PCR analysis of β-catenin mRNAs in ctrl and Yap-KO MC3T3 cells (mean ± SD, n = 3). j Co-immunoprecipitation analysis. The nuclear and cytoplasmic MC3T3 cell lysates were immunoprecipitated with anti-Yap (WH0010413M1, Sigma) and non-specific (nS) IgG. The resulting precipitates were subjected to immunoblot analysis using the indicated antibodies. Input, ~50 µg lysates. k, l Co-immunostaining analysis of β-catenin (C2206, Sigma) and YAP in ctrl and Yap-KO MC3T3 cells expressing YAP-GFP in the presence or absence of Wnt3A. k, representative images, Scale bar, 10 µm. l, quantification analysis (β-catenin fluorescence intensity in nuclei over cytoplasm by use of the NIH Image J software) (mean ± SD, n = 20 cells from 3 different experiments).
