Fig 7. Calvarial cells from Alk3f/fOsx-Cre mice show impaired osteoblastogenesis and bone mineralization in vitro, and BMSCs from Runx1f/fCol1α1-Cre or Runx2-/- mice show increased osteoblastogenesis and bone mineralization in vitro following overexpression of Atf4 or Runx1.
(A) Calvarial cells from newborn Alk3f/fOsx-Cre (ff/Δ), wild-type (f/f), and Osx-cre mice were submitted to osteoblastogenesis assays. Osteoblast differentiation was analyzed by ALP activity on day 14 or by von Kossa staining on day 21. (B) Western blot was performed to detect protein levels of Alk3, Runx1, Runx2, Osx, and Ocn on days 14 and 21. GAPDH is shown as a control. (C) ALP staining following retrovirus-mediated overexpression of Bmp7 in day 14 BMSCs from Runx1f/fCol1α1-Cre mice. (D) Western blot was performed to detect protein levels of Runx1, Bmp7, Alk3, Runx2, Atf4, Opn, Osx, and Ocn on day 14. GAPDH is shown as a control. (E) retrovirus-mediated overexpression of Atf4 in BMSCs from newborn Runx1f/fCol1α1-Cre (ff/Δ) or wild-type (f/f) mice. Osteoblast differentiation was analyzed by ALP activity on day 14. (F) Western blot was performed to detect protein levels of Runx1, Atf4, Bmp7, Alk3, Osx, Ocn, and Opn on day 14. GAPDH is shown as a control. (G) retrovirus-mediated overexpression of Runx1 in calvarial cells from newborn Runx2-/- or wild-type (+/+) mice. Osteoblast differentiation was analyzed by ALP activity on day 14. (H) Western blot was performed to detect protein levels of Runx2, Runx1, Opn, Atf4, Osx, and Ocn on day 14. GAPDH is shown as a control. Results are expressed as mean ± SD, n≧3 in each group. N.S, not significant *p < 0.05, **p < 0.01, ***p < 0.001.