Fig. 1
Expression of YAP in OB-lineage cells in culture and in vivo. a, b Western blot analysis of endogenous YAP levels in lysates of primary cultured BMSCs, OBs, and BMMs using the indicated antibodies (YAP, WH0010413M1, Sigma; β-catenin, Sigma, C7207). BMSCs and BMMs were derived from BM of mouse long bones at the indicated ages. OBs were in vitro differentiated from BMSCs at D14 cultures. The data were quantified by use of the NIH Image J software and presented in b (mean ± SD, n = 3 different cultures). *P < 0.05. c RT-PCR analysis of Yap gene expression in WT during OB differentiation. The data were present as mean ± SD (n = 5-different cultures). *P < 0.05. d Co-immunostaining analysis of YAP (1:200, mAb, WH0010413M1, Sigma) with β-catenin (1:2 000, pAb, C2206, Sigma) in BMSCs from WT mice (3-month old). e Immunostaining analysis of YAP (WH0010413M1, Sigma) in BMSCs from Ocn-Cre; Ai9 mice (1- and 3-month old). f–h Quantification analysis of data in d, e. The values of mean ± SD (n = 20) from three independent assays were presented. **P<0.01, ***P<0.000 1. i, j Immunohistochemical staining analysis of YAP/TAZ (pAb, #8418/D24E4, CST) in femur sections from 3-month-old Ocn-Cre;Ai9 mice. k Illustration of YAP expression in OB-lineage cells. In a–h, BMSCs were isolated from the indicated aged WT (c), Ocn-Cre; Ai9 (e–h), and CXCL12-dsRed mice (f). In i, j, the representative images are shown in i. The trabecular bone (Tb), cortical bone (Cb), growth plate (GP), and bone marrow (BM) are indicated. The YAP-tdTomato (Td) co-staining signals in OBs, osteocytes, lining cells, chondrocytes, and bone marrow cells were quantified [double-positive cells over total Td-positive cells in a selective region (%)] and presented in j (mean ± SD, n = 5 femur samples per genotype). *P < 0.05. Scale bar 20 µm.
