Fig. 6. Decreased bone resorption in Smad4 mutant mice. (A,B) Representative images of TRAP-stained distal femur from 8-week-old control (A) and Smad4 mutant mice (B). (C,D) Quantitative assessment by histomorphometric analysis of percentage of bone surface covered by mature osteoclasts (OcS/BS) and number of mature osteoclasts in bone perimeter (NOc/BPm) on the secondary spongiosa of distal femur in control (white bars) and Smad4 mutants (grey bars). All values are mean ± s.d. of eight mice. (E,F) Real-time PCR of TRAP and cathepsin K (CathK) from calvarial bone extracts of 7-week-old Smad4 mutant (grey bars) and control mice (white bars). (G-I) TRAP staining was performed following co-culture of osteoclast progenitor cells derived from the wild-type spleen with control (upper panel in G,H) or Smad4 mutant (lower panel in G,I) osteoblast-like cells generated from bone marrow mesenchymal stem cells. (J) RT-PCR analysis to detect transcripts of OPG, RANKL, Tgfb1, Bmp2 and Bmp4 in calvarial bones of 7-week-old Smad4 mutant and control mice. (K-N) Real-time PCR of OPG, RANKL, Tgfb1 and Bmp4 from calvarial bone extracts of 7-week-old Smad4 mutant (grey bars) and control mice (white bars) ^*P<0.05; ^**P<0.01. Bar, 100 µm (A,B).