3.3 NVP-BHG712 inhibited osteoclastogenesis and F-actin ring formation in mature osteoclasts at the early phase in vitro.
To further identify at which specific phase NVP-BHG712 exerts its inhibitory effect on osteoclastogenesis, RANKL-treated BMMs were incubated with NVP-BHG712 (0.4 μM) at different time points and for different durations (days 1-3, days 3-5, days 5-6, and days 1-6). TRAP staining illustrated that osteoclast differentiation was more strongly inhibited by NVP-BHG712 treatments in the first several days than in the later days, suggesting that NVP-BHG712 mainly suppressed osteoclast differentiation in the early phase (Fig. 3A, B). F-actin rings are considered the characteristic structure of mature osteoclasts. To further determine the inhibitory effect of NVP-BHG712 on osteoclast function, immunofluorescence staining of the F-actin ring was performed in BMMs that were exposed to NVP-BHG712 treatments. Similar to the prior inhibitory effects, NVP-BHG712 more significantly inhibited the formation of F-actin ring structures by mature osteoclasts in the first several days than in the later days, as demonstrated by a decrease in F-actin numbers compared to total osteoclast numbers (Fig. 3C, D). In conclusion, the results suggest that osteoclast function is mainly inhibited by NVP-BHG712 in the early phase.