Research Paper Volume 12, Issue 9 pp 8652—8668

Figure 4. Effects of CDH-13 on RANKL-induced osteoclast differentiation. (A) BMMs were cultured for three days in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with one of the candidate proteins (ANTXR2, CDH-13, CD163, COMP, DKK3, periostin or secretogranin-1; 100 ng/mL). Osteoclasts were stained with TRAP. (B) BMMs were incubated with various concentrations of CDH-13 (0, 1, 10 and 100 ng/mL). (C) TRAP-positive multinucleated cells with more than five nuclei were counted. (D) M-CSF-treated BMMs were pretreated with CDH-13 or the vehicle for 30 min. RANKL (100 ng/mL) was used to stimulate the cells at the indicated times, and immunoblotting was used to detect members of the RANKL/mitogen-activated protein kinase and NF-κB signaling pathways. (E, F) Differentiated osteoclasts were cultured in the presence of the vehicle or CDH-13 (1, 10 or 100 ng/mL) on dentin slices. Resorption pits were visualized with hematoxylin, and the resorption areas were measured. Error bars represent ± SEM. ** P< 0.01, * P < 0.05; NS, not significant.