Fig. 3. Silencing of Sox4 mRNA in WT primary osteoblast cultures with siRNA. (A) Real-time RT-PCR analyses of mRNA levels in siRNA-treated osteoblasts expressed relative to osteoblasts treated with control (scrambled) siRNA (set to 1, broken line), and normalized to GAPDH mRNA. Osx, osterix; OCN, osteocalcin; Coll1A2, collagen1A2; ALP, alkaline phosphatase; Runx2; OPN, osteopontin; PTHR1, PTH/PTHrP-receptor-1; PTHrP, PTH-related peptide. (B) Incorporation of [³H]thymidine in proliferating WT osteoblasts following treatment with control or Sox4 siRNA, respectively. (C) Photomicrographs of WT osteoblast cultures treated with control or Sox4 siRNA, respectively, stained for ALP activity. (D) Quantification of ALP-positive cells per well in C. (E) Biochemical activity of ALP in siRNA-treated osteoblasts. (F) mRNA levels of Runx2, OCN and Sox4 following treatment of WT cells with specific Runx2 siRNA compared with control siRNA, quantified by real-time RT-PCR and related to GAPDH mRNA as control (set to 1, broken line). In A,B,D-F: ^***P<0.001, ^*P<0.05 versus siRNA control (ctrl); bars, mean ± s.d. of triplicates (n=2).