Fig. 4.

Less inhibitory effects of DL on the osteoclast differentiation and NFATc1 expression in Nrf2-deficient cells. (A-C) Wild-type and Nrf2-knockout BMMs were incubated with RANKL in the presence of the indicated concentrations of DL for 4 days (A) or 2 days (B), and in the presence of 1.5 μM DL for 2 days (C). The cells were fixed and stained with TRAP. Scale bar, 200 μm. TRAP-positive multinucleated cells containing more than three nuclei were counted and the percentage of osteoclast formation was plotted as a function of the concentration of DL (A). The amount of NFATc1 protein was analyzed by immunoblotting (B) and the expression of NFATc1 and its target gene was assessed by real-time PCR (C). All values represent means ± SD. N = 3. ***P < 0.001 between the indicated groups. (D) Proposed mechanism of DL inhibition of RANKL-induced NFATc1 expression and osteoclast differentiation.