y in the medium of BMM cultures in the presence of RANKL and M-CSF. E, Viability was determined by MTT assay after 3 days. Data are expressed as means SD of three experiments. P < 0.01 vs. untreated controls. doi:10.1371/journal.pone.0130174.g001 multinucleated cell formation from mouse BMMs. In this study, we investigated the effects of rhinacanthin C on osteoclast differentiation and bone resorption pit formation. Rhinacanthin C produced dose-dependent inhibition of RANKL-induced TRAP-positive multinuclear osteoclast formation and TRAP activity in BMM culture . In our previous study, rhinacanthin C exhibited non-apoptotic cytotoxicity against tumor cells. To investigate whether osteoclastogenesis inhibition by rhinacanthin C is due to its cytotoxicity, we measured the viability of RANKL-induced osteoclasts exposed to rhinacanthin C. Fig 1E shows that rhinacanthin C did not suppress viability even at 2.0 M, at which concentration it abolished RANKL-stimulated osteoclast formation. Thus, the inhibitory effects of rhinacanthin C on osteoclastogenesis seemed not to be due to cytotoxicity. Osteoclasts differentiated from the monocyte-macrophage lineage. Therefore, we examined the effects of rhinacanthin C on macrophage colony formation with M-CSF from BMCs. The 5 / 17 Rhinacanthin C Suppresses Osteoclastogenesis macrophage-type colonies formed from BMCs did not differ in the absence or presence of rhinacanthin C. Furthermore, the numbers of zymosan-incorporated phagocytic macrophages did not significantly differ in the presence of absence of rhinacanthin C. Thus, macrophage formation and phagocytic function stimulated by M-CSF are not inhibited by rhinacanthin C. We further characterized the effects of rhinacanthin C on osteoclastogenesis by performing two experiments. First, BMCs were cultured with rhinacanthin C for 3 days in the presence of M-CSF, followed by RANKL plus M-CSF to induce osteoclast formation. In the second experiment, BMCs were cultured with rhinacanthin C and then the BMMs were cultured with RANKL plus M-CSF, also in the presence of rhinacanthin C. In experiment 1, RANKL-stimulated osteoclast formation remained essentially the same, regardless of pretreatment with rhinacanthin C. However, after RANKL stimulation, rhinacanthin C provided dose-dependent inhibition of osteoclast formation, as shown in experiment 2. These results suggest the inhibitory role of rhinacanthin C in osteoclastogenesis does not occur at the stage macrophage formation and function as an osteoclast progenitor but at the stage of RANKL-stimulated osteoclast formation step. Next, we sought to identify what stage of osteoclast differentiation is more strongly affected PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19705070 by rhinacanthin C. Rhinacanthin C was added at various stages of osteoclastogenesis over 3 days. Late addition of rhinacanthin C had little effect on TRAP activity and TRAP positive-multinucleated cell formation. Treatment on days 1 and 2 resulted in a significantly lower number of TRAP positive-multinucleated cells versus treatment on days 2 and 3, indicating that rhinacanthin C suppressed early-to-middle stage RANKL-stimulated osteoclastogenesis PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19703425 form BMMs. The numbers of TRAP-positive multinucleated cells and TRAP activity were higher in cultures treated on day 1 than in those treated on day 2. These results suggest the inhibitory effects of rhinacanthin C is reversible, and differentiation potential of 
BMMs treated with rhinacanthin C on day 1 is recovered over 2 days culture PF-562271 without r