Fig. 2. Primary cultures of calvarial osteoblasts derived from Sox4^+/– and WT mice. (A) Real-time PCR analyses of osteoblast characteristic mRNAs extracted from Sox4^+/– and WT primary osteoblast cultures: Osx, osterix; OCN, osteocalcin; Coll1A2, collagen1A2; ALP, alkaline phosphatase; Runx2; BMP-2, bone morphogenetic protein-2; OPN, osteopontin; PTHR1, PTH/PTHrP-receptor-1; PTHrP, PTH-related peptide. Results (from three independent experiments) were normalized to -actin or GAPDH, and expression in Sox4^+/– cells is presented relative to WT (set to 1, broken line). (B) Representative immunoblot of lysates from WT and Sox4^+/– osteoblasts, using anti-Sox4 and anti-actin antibodies as described in the Materials and Methods. (C) Representative micrographs of ALP histochemical staining (20x magnification) in WT and Sox4^+/– osteoblasts cultured for 7 days (n=4). (D) Biochemical quantification of ALP activity in the osteoblast cultures shown in C. (E) Micrographs of von Kossa-stained osteoblast cultures (20x magnification) demonstrating mineralization (dark areas) of nodules after 3 weeks of culture in medium containing ascorbic acid and -glycerophosphate. (F) Densitometric quantification of von Kossa-stained mineralized nodules expressed as per cent of WT staining. In each of three independent experiments, 4-10 representative microscopic fields were examined. (G) Incorporation of [³H]thymidine in proliferating osteoblasts. Data from two independent experiments were normalized and compared with mean WT values (n=24 for each genotype). Bars, mean ± s.d. ^***P<0.001, ^*P<0.05, unpaired Student's t-test (with Welch correction in F).