Effect of WEAO on RANKL-induced osteoclast differentiation in BMMs. (A, B) BMMs were cultured with vehicle (distilled water) or WEAO (10–160 μg/mL) in the presence of M-CSF (60 ng/mL) and RANKL (100 ng/mL) for 4 days. (A) Cells were fixed and stained for TRAP activity. Scale bar, 200 μm. (B) The number of osteoclasts (Oc) was counted. (C) The viability of BMMs was determined by Cell Counting Kit-8 assay. (D) BMMs were cultured in the presence of M-CSF and RANKL for 4 days, and WEAO (80 μg/mL) was added to the BMM cultures at the indicated days. The number of osteoclasts was counted on day 4. (E) HPLC chromatograms of WEAO and a standard mixture of chrysin, tectochrysin, and nootkatone at 203 nm. (F) BMMs were cultured with vehicle (dimethylsulfoxide) or nootkatone (10–40 μM) in the presence of M-CSF and RANKL for 4 days, and the number of osteoclasts was counted. (G) The effect of nootkatone on viability of BMMs. *p < 0.05; **p < 0.01 versus vehicle-treated control.