Figure 6.

JN-2 inhibits osteoclast differentiation indirectly. (a) BMMs were cultured in the presence of M-CSF (60 ng ml⁻¹) and RANKL (RL; 100 ng ml⁻¹) with or without CM+DMSO or CM+JN-2 (10 μM) for 4 days. Differentiated cells were stained for TRAP, and cells containing 3 or more nuclei were counted. Scale bar is 100 μm. (b) Mouse-calvarial osteoblasts were cultured with DMSO or JN-2 (10 μM) in the presence of PBS or CXCL10 (300 ng ml⁻¹). After 24 h of incubation, RANKL and OPG mRNA levels were analyzed by real-time PCR. (c) BMMs and mouse-calvarial osteoblasts were co-cultured for 9 days with or without CM from 4T1 cells treated with DMSO or JN-2 (10 μM). The cells were stained for TRAP, and cells containing three or more nuclei were counted. Scale bar is 100 μm (*P<0.05). (d) Mouse-calvarial osteoblasts were cultured for 24 h with or without CM from 4T1 cells treated with DMSO or JN-2. RANKL and OPG mRNA levels were analyzed by real-time PCR (*P<0.05).