Figure 4.

Samd4 (sterile alpha motif domain containing protein 4) affected osteoblastogenesis in vitro. (a) Representative photographs of fast blue staining (top, cultured in OBD for 7 days) and alizarin red staining (bottom, cultured in OBD for 14 days) of differentiated osteoblasts wild-type (WT) and Samd4^PB/PB mice. (b, c) Quantitative parameters of Alp activity (b) and alizarin red staining (c) analyzed by colorimetric assay. (d–f) Analysis of the transcript levels of the indicated genes by quantitative PCR using RNA isolated from primary osteoblasts of WT and Samd4^PB/PB mice. (g) The representative photographs of fast blue staining and alizarin red staining of primary osteoblasts infected with green fluorescent protein (GFP)- and Samd4-expressing lentivirus. (h, i) Quantitative parameters of Alp activity staining (h) and alizarin red staining (i) analyzed by colorimetric assay. (j) The representative photographs of fast blue staining of osteoblasts differentiated from Samd4^PB/PB osteoblasts infected with lentivrus-expressing GFP, Samd4 or two Samd4 mutants. (k–m) Analysis of the transcript levels of the indicated genes by quantitative PCR. Values represent mean±s.d. (n=6 (b, c, h and i) and 3 (d–f and k–m) for each genotype). P-values were obtained from t-tests with paired or unpaired samples, *P<0.05, **P<0.01. Bars=2 mm in panels (a, g and j).