Fig. 3
Reductions in OB differentiation and function and increases in adipocyte differentiation in YapOcn-Cre-BMSC/OB cultures. a, b In vitro OB differentiation was decreased in YapOcn-Cre-BMSC cultures. ALP staining images at D14 cultures are shown in a, and ALP activity (ALP-positive area/over total area, normalized by three ctrl cultures) is presented in b. c, d In vitro OB function (viewed by Alizarin Red staining) was attenuated in YapOcn-Cre-OB cultures. At D28 of BMSC/OB cultures, cells were stained for Alizarin Red S (c), and the data were quantified and illustrated in d. In b, d, the values of mean ± SD (n = 3 different cultures) are presented. **P<0.01. e–h Both CFU-F and CFU-OB were reduced in YapOcn-Cre-BMSC cultures. CFU-F and CFU-OB assays are described in the supplemental methods. The Crystal Violet (e) and Alizarin Red S (g) staining images and their quantifications (mean ± SD, n = 3 different cultures) are shown. i, j Oil Red O staining analysis showed an increase in bone marrow fat-containing cells in YapOcn-Cre mice (3-month old). i, representative images and j, quantification analysis of bone marrow adipocytes over total bone marrow cells in femur mid diaphysis. (mean ± SD, n = 5 mice/genotype, male). **P<0.01. k, l BMSCs from ctrl (Yapf/f) and YapOcn-Cre mice (3-month old) were induced for adipocyte (AD) differentiation, and at D21 cultures, they were stained with Oil Red O. k, adipocyte images; and l, quantification analysis (mean ± SD, n = 3 different experiments). Scale bar, 100 µm, ***P<0.000 1. m–o Real-time PCR analyses of the indicated genes’ expression in ctrl and YapOcn-Cre BMSCs, OBs (D14), and ADs (D21) (mean ± SD, n = 3 different assays). *P < 0.05.
