Fig. 4. Effects of SPA0355 on RANKL-induced osteoclast differentiation.

(A) BMMs (1×10⁴) were treated with the indicated concentrations of SPA0355 for four days, and cell viability was determined by MTT assay (n=5). (B) BMMs were cultured for four days with M-CSF (10 ng/ml) and sRANKL (30 ng/ml) in the presence or absence of 10 and 20 µM SPA0355. For ICI 182,780 treatment, 100 nM compound was used 2 h prior to SPA0355 treatment. Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP solution. TRAP-positive multinucleate cells with three or more nuclei were scored as osteoclasts (n=4). ICI, ICI 182,780. (C) The mRNA expression of osteoclastogenesis-related genes was analyzed by real-time RT-PCR (n=10–12). Values are the mean±SEM. ^*p<0.05 and ^**p<0.01 vs. untreated control. ^#p<0.05 and ^##p<0.01 vs. RANKL+VEH.