Fig. 4

Declines of cell proliferation and OB-progenitor cells in Yap^Ocn-Cre mice. a, b Reductions in OB-progenitor cell proliferation in Yap^Ocn-Cre BMSC culture. BMSCs were incubated with BrdU (10 μmol·L^-1, 2 h) and then subjected to anti-BrdU immunostaining analysis. a, representative images, Scale bar, 20 µm. b, BrdU⁺ Td⁺ cells over total Td⁺ cells (mean ± SD, n = 50 from 3 different cultures), *P<0.05. c–f Decreases in Td and β-catenin double-positive BMSCs from Yap^Ocn-Cre mice. BMSCs from 3-month-old ctrl (Ocn-Cre;Ai9) and Yap-CKO (Ocn-Cre;Ail9;Yap^f/f) mice were subjected to co-immunostaining analyses using the indicated antibodies (YAP, WH0010413M1, Sigma; β-catenin, Sigma, C2206). c, representative images, Scale bar, 20 µm. d–f, quantification analyses (mean ± SD, n = 50 from 3 different cultures), ***P<0.000 1. g–j FACS analysis of Td⁺ cells in cultured BMSCs (g, h) and isolated total bone marrow cells (BMCs) (I, j) from 3-month-old WT, Ocn-Cre;Ai9, and Ocn-Cre;Ai9;Yap^f/f mice. Representative FACS data were shown in g, i. The quantification analyses were presented in h, j. (Mean ± SD, n = 3 from 3 different cultures or mice. **P < 0.01). k–n Analysis of BrdU⁺ proliferative cells in ctrl and Yap^Ocn-Cre femur bone sections. BrdU (100 μg·kg^-1) was injected (i.p. four times over a 12 h period at 0, 4, 8, 12 h) into ctrl and Yap^Ocn-Cre mice (3 months), and 12 h after the last injection, mice were sacrificed (k), and their femur bone sections were subjected to anti-BrdU antibody immunostaining analysis. l–m, representative images; n, quantification analysis of BrdU⁺ cell density in Tb (trabecular bone), Ec (endocortical bone surface), and Ps (periosteum) (Mean ± SD, n = 3 male mice. *P < 0.05).