Figure 4.

Differentiation of primary calvarial osteoblasts in neonatal D2J and D2J/Gpnmb⁺ mice. A: Western blots of OA glycoprotein in primary osteoblasts cultured for 7 days. B: ELISA of OA protein levels in osteoblast cell lysates, normalized to total protein concentration. C: Macro- and microscopic images of 14-day differentiated osteoblasts stained for ALP (asterisks). D: ALP activity in osteoblast matrix and cell lysates. E: Macro- and microscopic images of 14-day differentiated cultures stained for collagen (asterisks) with Sirius Red in D2J and D2J/Gpnmb⁺. F: Quantification of Sirius Red collagen staining in D2J and D2J/Gpnmb⁺ cultures. G: Percentage area fraction of Sirius Red–stained matrix in D2J and D2J/Gpnmb⁺ cultures. Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01. Scale bar = 100 μm.