Fig. 1.

Inhibition of RANKL-induced NF-κB activation by DL. (A) RAW264.7 cells were transfected for 24 h with 0.45 μg of pNF-κB-Luc (NF-κB reporter plasmid) and 0.15 μg of pRL-SV40 (internal control). The cells were treated with RANKL for 24 h in the presence of 1.5 μM DL. The luciferase activity of each cell lysate was measured using a dual-luciferase assay system. The activity of firefly luciferase was normalized to that of the Renilla enzyme and expressed as fold increase relative to the activity of RANKL-untreated cells. (B-E) BMMs were incubated with RANKL and M-CSF in the presence of 1.5 μM DL for 24 h (B), the indicated times (C, D) or 15 min (E). The mRNA levels of individual genes were assessed by real-time PCR and presented as fold induction (B). Cell lysates were subjected to immunoblotting analysis (C, D). The cells were stained with p65 antibody and DAPI, and then photographed under a fluorescence microscope. Scale bar, 20 mm (E). All values represent means ± SD. N = 3. ***P < 0.001 between the indicated groups.