Figure 4.

Effect of PTH on differentiation of primary calvarial osteoblasts isolated from the WT and the pd mice. (A) Representative von Kossa staining of primary calvarial osteoblasts isolated from the WT and the pd mice. The cells were differentiated with ascorbic acid and β-glycerophosphate for 19 days with or without 100 nM PTH. Following von Kossa staining, mineralized bone nodules of phosphate deposits were counted. The results are expressed as percent relative expression of bone nodules without PTH treatment. Results from representative culture plates are shown. (B) Primary calvarial osteoblasts isolated from the WT and the pd mice were differentiated for 5 days as above, with and without PTH. Results from representative ALKP staining of 5 day differentiated cultures are shown. ALKP activity was measured in duplicate plates, and the results are expressed as fold change with respect to untreated cells. (C) Real-time PCR analysis was performed to analyze RNA extracted from differentiated WT and pd calvarial osteoblasts with and without PTH treatment. GAPDH was used as an internal control. Results are expressed as mean±S.D. from five to six independent experiments. a, P<0.001. P, PTH; pd, phosphorylation-deficient PTH1R; WT, wild-type; N, undifferentiated control.