SDX-101 and SDX-308 inhibit RANKL-induced osteoclast formation and NF-κB activation in RAW 264.7 cells. (A) RAW cells (1 × 10⁴ per well) were cultured in the presence of RANKL (50 ng/mL), and drug vehicle, SDX-101, or SDX-308 was added to appropriate wells. After 5 days, the cells were fixed and stained for TRAP activity. TRAP⁺ multinucleated (> 3 nuclei) cells were recorded for each well. Data are the mean ± SD of at least 3 measurements. (B) RAW cells, transiently transfected with the 3kB-Luc-SV40 reporter gene, were treated with SDX-101, SDX-308, or vehicle in the presence or absence of RANKL. Luciferase activity was determined 8 hours after RANKL stimulation (150 ng/mL). *Significant difference from RNAKL-treated only (P < .05). (C-D) Raw cells were incubated with drug vehicle, SDX-101, or SDX-308 for 1 hour, treated with RANKL (100 ng/mL) for 30 minutes, and then lysed. Nuclear extracts (NE) and cytoplasmic extracts (CE) were prepared using a commercial kit (Pierce, Rockford, IL). Phospho-p65 in CE and p65 in NE were detected by Western blot assay. (E) Phospho-IκB-α was detected in whole cell lysates of RAW cells by Western blot analysis. (C-E) β-Actin served as loading control.