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Fig. 1. Targeted disruption of Smad4 in mouse osteoblasts. (A-I) Characterization of Cre activity in 6-week-old OC-Cre;ROSA26 double transgenic mice. LacZ staining was detected in osteoblasts on trabecular bone (A), under the periosteum (B) and in osteocytes embedded in cortical bone (C). (D-I) Primary bone marrow cells of OC-Cre/ROSA26 double heterozygous (D-F) and ROSA26 (G-I) mice cultured for 12 days were subjected to LacZ staining (D,E,G,H) or Alizarin Red staining (F,I). E and H were higher magnification of areas in D and G, respectively. (J) Southern blot showing Cre-mediated recombination in bone tissues. The 4.3 kb fragment stems from the Smad4 conditional allele, the 7.2 kb fragment stems from the Smad4 allele after Cre-mediated recombination in osteoblasts. (K) Southern, using the probe previously described (Zhang et al., 2005), blot of Smad4 mutant calvarial genomic DNA at different ages. (L-M) Blockage of BMP and TGFβ signaling. Primary Smad4^Co/Co osteoblasts were transfected by pCMV2 and pCMV2-Cre vector together with BMP reporter construct, 12xSBE-OC-Luc (L) or TGFβ reporter construct, 3TP-Lux (M), and then treated with 50 ng/ml BMP-2 (L) or 5 ng/ml TGFβ1 (M). Inactivation of Smad4 blocked luciferase activity stimulated by BMP-2 and TGFβ1. ^*P<0.01. Bar, 25 µm (A-C).

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