Inhibition of RANKL-induced osteoclast differentiation and NFATc1 expression by BA. BMMs were cultured in the presence of M-CSF and were treated with RANKL in the presence of the indicated concentrations of BA for 3 and 4 days (A) and (B), 4 days (C) or 2 days (E), and in the presence of 10 μmol/L BA for 2 days (G), 4 days (D) or the indicated times (F). (A) The cells were fixed, subjected to TRAP staining and visualized under a light microscope. Scale bar, 200 μm. (B) TRAP-positive multinucleated cells (MNCs) were counted. ***P < 0.001 control vs. BA. (C) The cell viability assay was performed using the WST-1 reagent. The percentage of viable cells was calculated based on the absorbance of the sample relative to positive control at 450 nm. M, M-CSF only; M + R, M-CSF plus RANKL. *P < 0.05, ***P < 0.001 control vs. BA. (D) The cells were stained with Alexa Fluor 488-phalloidin and DAPI, and then photographed under a fluorescence microscope. Scale bar, 200 μm. (E) and (F) The expression of NFATc1 was analyzed by immunoblotting. (G) Real-time PCR was performed to quantify the mRNA levels of Nfatc1 and its target genes. Relative levels of individual mRNA were normalized to those of β-actin mRNA and presented as fold induction. Veh, vehicle. ***P < 0.001 between the indicated groups.