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. 2014 Oct 30;289(52):35868–35881. doi: 10.1074/jbc.M114.589614

FIGURE 3.

FIGURE 3.

Inhibition of OC differentiation, fusion, and survival by the truncated OSTM1 treatment. A, schematic illustration of the murine Ostm1 cDNA structure and the OSTM1 expression plasmids. Black box, FLAG epitope (Flag); dotted box, Fc fragment of human IgG1 (hFc). B, purification of recombinant OSTM1ΔTM-hFc protein. C, cell surface binding assay with the OSTM1ΔTM-hFc protein. The cell surface binding of OSTM1ΔTM-hFc (thick solid lines) or hFc protein (dotted lines) in various cells was determined through flow cytometry using a mouse anti-FLAG primary antibody and a PE-labeled anti-mouse IgG secondary antibody. Cells treated with the PE-labeled anti-mouse IgG secondary antibody alone were used as a control (mock; gray with thin solid lines). D, inhibitory effects of truncated OSTM1 (OSTM1ΔTM-hFc) on OC differentiation. The number of TRAP+ MNCs (≥3 nuclei) was counted from the BMOC formation assay (left panels), the Raw264.7-derived OC-like cell formation assay (middle panels), and the co-culture assay (right panels). E, OSTM1ΔTM-hFc inhibits the fusion of preOCs. The fusion of TRAP+ mononuclear preOCs was monitored through TRAP staining (left panels; original magnification, ×100). The number of MNCs derived from the fusion of preOCs was counted (right panel). F, OSTM1ΔTM-hFc inhibits the survival of OCs. Apoptosis of BMOCs was induced through the depletion of RANKL and M-CSF for the indicated times. The survival of OCs was monitored through TRAP staining (left panels; original magnification, ×100). TRAP+ MNCs (≥3 nuclei) were counted, and the relative survival rate of OCs after OSTM1ΔTM-hFc treatment was calculated (right). G, effects of heat inactivation of the truncated OSTM1 on OC differentiation. BM cells were differentiated into BMOCs using M-CSF and RANKL in the presence of heat-inactivated proteins (12.5 μg/ml). OCs were analyzed and photographed (top panels; original magnification, ×100) after TRAP staining. The TRAP solution assay was performed (bottom left). The number of TRAP+ MNCs (≥3 nuclei) was counted (bottom right). NS, not significant. *, p < 0.05; **, p < 0.01. Error bars, S.D.