Figure 1. XN suppresses human and mouse osteoclast differentiation.

(A) The effect of xanthohumol (XN) on mouse BMMs differentiation. BMMs were incubated with M-CSF (20 ng/ml) and RANKL (30 ng/ml), followed by addition of different concentrations of XN for 6 days. The cells were stained for TRAP assay and photographed (×40; left). The numbers of TRAP positive multinucleated (>5 nuclei) osteoclasts were counted (right). (B) The effect of XN on RAW264.7 cell differentiation. RAW264.7 cells were treated with RANKL (30 ng/ml) and different doses of XN for 3 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (C) The effect of XN on human PBMC differentiation. PBMCs (5 × 10⁵ cells) were stimulated with hRANKL (50 ng/mL) and hCSF1 (20 ng/mL) for 8–9 days. The cells were stained for TRAP assay and photographed (×40; left) and the numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). (D) XN inhibits RANKL-induced osteoclast differentiation at the early stage. Osteoclast precursor BMMs were cultured with M-CSF and RANKL for differentiation into mature osteoclasts in 6 days. XN (5 μM) were added at indicated time (day). The cells were fixed and stained for TRAP activity. (E) The effect of XN on cell viability in BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with different concentrations of XN for 5 days, and the cell viability was measured by SRB assay. (F) The effect of XN on cell viability in human PBMC cells. Human PBMC cells were incubated with hCSF1 (20 ng/mL) and different concentrations of XN for 4 days. The cell viability was measured by SRB assay. Column, means of experiments performed in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001. N.S., no significant.