es, and then immunoblotted with primary antibody. The blots were incubated with horseradish peroxidase-conjugated secondary 
antibodies for 1 h, and chemiluminescence was detected with an Immobilon system. For immunoprecipitation, cell lysates extracted with RIPA I buffer were incubated with anti-TRAF6 or anti-TAK1 antibody for 2 h at 4C, followed by 1-h 3 / 17 Rhinacanthin C Suppresses Osteoclastogenesis incubation at 4C with protein G-Sepharose beads. After washing with RIPA II buffer, immune complexes were analyzed by western blotting. Protein band intensities were quantified using ImageJ. Cell viability Viability was evaluated using a cell proliferation assay kit. At the end of culture, a 1/10 volume of reagent was added and incubated for 2 h. Absorbance was measured at 492 nm. RANKL and endotoxin-induced bone loss in vivo Eight-week-old male mice were divided into three groups. Vehicle or RANKL with or without rhinacanthin C was injected daily into the subcutaneous tissue overlying the calvaria under anesthesia with pentobarbital. Eight-week-old normal male or osteoprotegerin knockout mice were used for the LPS experiments. Vehicle or lipopolysaccharide with or without rhinacanthin C was injected. The mice were sacrificed on day 5. Calvariae were dissected, fixed in 4% paraformaldehyde, and stained for TRAP Birinapant biological activity activity. Three-dimensional reconstruction images of calvarial bone were obtained by micro-focal computed tomography scanning at the Research Institute of Bone Structure Analysis. The X-ray tube was operated at an acceleration voltage of 60 kV and current of 100 A. The geometry of the setup and resulting constraints in resolution lead to a voxel size of 32 32 32 m. The 3-D images were analyzed using Tri-3D Bon software. Trabecular bone volume, total tissue volume, bone volume fraction were calculated in 3D mode. Static bone morphometric parameters trabecular separation, and trabecular bone pattern factor were also calculated. Ethics Statement This study was performed according to the Fundamental Guidelines of the Ministry of Education, Culture, Sports, Science and Technology of Japan for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions. The Animal Care and Use Committee of Meikai University approved all animal PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19704080 experiments. All animal experiments were performed under anesthesia to limit pain. The mice were sacrificed by spinal cord dislocation under anesthesia with pentobarbital. Statistical analysis Data were analyzed by ANOVA and are presented as the means SD of at least three independent experiments. In all analyses, P < 0.05 was taken to indicate statistical significance. Results Rhinacanthin C inhibits RANKL-mediated osteoclast formation from mouse BMMs without cytotoxicity Rhinacanthin C is a naphthoquinone derivative containing an alkyl side chain. We previously reported that rhinacanthin C is a strong inhibitor of RANKL-stimulated TRAP-positive 4 / 17 Rhinacanthin C Suppresses Osteoclastogenesis Fig 1. Effects of rhinacanthin C on osteoclastogenesis and cell viability in BMM cultures. A, Chemical structure of rhinacanthin C. B, BMCs were cultured for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19703425 3 days with M-CSF, then with M-CSF alone or M-CSF plus RANKL in the presence or absence of rhinacanthin C. The cells were stained for TRAP activity. Bar, 100 m. C, Dose-dependent effects of rhinacanthin C on RANKL-induced TRAP-positive multi-nuclear cell formation from BMCs. D, Dose-dependent rhinacanthin C inhibition of TRAP activit