Fig. 5.

The reciprocal effects of CSE on bone remodeling mediated by RANKL-dependent pathway. a Non-adherent bone morrow cells collected from Fvb/n mice were placed into 96-well plates and treated with M-CSF and RANKL in the presence or absence of CSE at indicated concentrations for 7 days, then stained for TRAP. (Left) TRAP-positive multinucleated cells indicate osteoclast formation. (Right) Quantification of TRAP-positive multinucleated cells per well. CSE dose-dependently increased the number of osteoclast-like multinucleated cells. b CSE treatments induced osteoclast-specific gene expression in a dose-dependent manner. Samples were evaluated in quadruplicate. Results are reported as mean ± SD. c BMSCs and MC-4 cells were placed in 6- or 12-well plates and cultured in differentiation medium with the indicated concentrations of CSE for 10 days, and inorganic phosphate (5 mM) was added to the culture medium for the last 4 days. The capacity of bone mineralization was measured by von Kossa staining. CSE dose-dependently inhibited bone mineralization in both MC3T3 cells and BMSCs. d Total RNA was extracted from differentiated BMSCs, and RANKL and OPG gene expression were determined by real-time RT-PCR. The ratio of OPG to RANKL expression was dramatically reduced by CSE. All the data are from three independent experiments