Figure 3.

Bone marrow cells from CD97 KO mice have reduced ability to form osteoclasts and reduced resorption activity. Bone marrow cells from WT and CD97 KO mice were cultured with M-CSF and RANKL (30 ng/ml) for duration as indicated. A) Representative micrographs of cultures for 4 days. B) Bone marrow cells from WT and CD97 KO mice were cultured for up to 5 days in the presence of M-CSF and RANKL. TRAP(+) OCL per well was scored. C) BMM cells from WT or CD97 KO mice were cultured for 5 days and the size of the osteoclasts were measured using the CellSens image analyzing software. D) Attenuation of Sirp-1α protein expression in BMM cultures from CD97-deficient mice. E) Cell proliferation assay was performed using WST-1 reagent. BMM cells were plated in a 96 well plate and treated with M-CSF and RANKL for up to 5 days prior to the assay. F) Bone marrow cells from WT and CD97 KO mice were cultured with M-CSF and RANKL on collagen gels and an equal number of fully differentiated multinucleated cells were seeded on bone slices for 24 hrs and the area of resorbed pit per osteoclast was measured. CD97 KO mice have decreased osteoclast precursors. G) CFU-GM assay. Bone marrow cells from WT and CD97 KO mice were cultured in semisolid methylcellulose to examine the number of osteoclast precursor cells in the presence GM-CSF for 7 days and the number of colonies containing more than 40 cells were counted. H) P4 (TN c-fms[CD115]^hic-kit[CD117]^hi) population in total bone marrow cells of WT and CD97 KO mice were measured by flow cytometry. I) FACS sorted cell (TN c-fms⁺) from long bones (femurs and tibia) were cultured with M-CSF and RANKL (30 ng/ml) for 5 days prior to TRAP(+) staining. Experiments were performed at least three times and the representative experiment is shown. TN: B220⁻CD3⁻CD11b^−/lo. Values represent mean±SEM. *, Significant effect of CD97 KO mice, p<0.05.