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.