Figure 3.

AaE suppresses RANKL-induced osteoclastogenesis and the function of mature osteoclasts. (a) BMMs were cultured in α-MEM containing M-CSF (30 ng/mL) and AaE at the indicated concentrations for 5 days. Cell viability was measured with an MTT assay. The data are expressed as the mean ± SE of triplicate experiments. ^* P < 0.05, ^** P < 0.01 versus BMMs without AaE. (b) BMMs were incubated in α-MEM with M-CSF (30 ng/mL), RANKL (100 ng/mL), and the indicated concentrations of AaE for 5 days. The differentiated osteoclasts were detected by TRAP staining, and TRAP-positive multinucleated cells with more than 3 nuclei were counted using light microscopy (magnification, ×100). (c) The BMMs were cultured in α-MEM containing M-CSF (30 ng/mL) and RANKL (100 ng/mL) onto Osteo Assay Surface Plates for 5 days. The cells were then treated with the indicated concentrations of AaE for an additional 2 days. The resorption pits (*) were observed using light microscopy (magnification, ×100), and the resorbed area was calculated using ImageJ software. (d) MMP-2 and MMP-9 activities in the culture media of osteoclasts were detected by gelatin zymography as described in the Methods section. (e) Cathepsin K activity in the culture media and lysates of the osteoclasts was measured using a commercially available cathepsin K assay kit according to the manufacturer’s instructions. The images are representative, and the data are expressed as the mean ± SE of triplicate experiments. ^# P < 0.01 versus BMMs without RANKL (C), ^* P < 0.05, ^** P < 0.01 versus BMMs with RANKL alone.