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Osteoblast differentiation of MC3T3-E1 cells. MC3T3-E1 cells were plated at a density of 1 × 105 cells/mL on 6-well plates. After 24 h, at about 80% confluency, the cells were transfected with 100 pmol of Foxc2-siRNA or control-siRNA using a lipofectamine plus reagent. Induction of differentiation was achieved by treatment with the standard induction mixture containing β-glycerophosphate (10 mM), ascorbic GSK1349572 (50 μg/mL), and dexamethasone (10 nM) on 1 day post-confluent cells.
ALP and Alizarin red staining in vitro. Cells were washed twice with PBS, fixed with 2% paraformaldehyde, and stained for ALP according to the manufacturer’s instructions (Sigma). For Alizarin red staining, cell were fixed in 10% formalin/PBS and stained with 2% Alizarin red S (pH 4.0) solution.
Results
Effect of Foxc2 on mesenchymal cell differentiation in calvaria organ culture
To determine the role of Foxc2 in osteoblast differentiation of suture mesenchymal cells, we examined ALP and BSP expression after calvaria organ cultures (Fig. 2). ALP expression was observed in suture mesenchymal cells in the Foxc2 overexpressed calvaria whereas ALP was weakly expressed in suture mesenchymal cells in the control (Fig. 2C and D). Moreover, we found that BSP, a marker for mature osteoblasts, was strongly expressed in suture mesenchymal cells in the Foxc2 overexpressed calvaria as shown by the arrow (Fig. 2H). This finding indicated that mesenchymal cells in the suture mesenchyme were differentiating into mature osteoblasts. There was weak BSP staining in suture mesenchymal cells of the control as shown by the arrow (Fig. 2G).