Figure 2.

Britanin suppresses the expression of osteoclast and Nrf2-associated markers during osteoclastogenesis. (A-E) BMMs were cultured with M-CSF (10 ng/ml) and RANKL (20 ng/ml) in the absence or presence of britanin for 4 days and RT-qPCR was performed to analyze the expression of (A) Nfatc1, (B) Ctsk, (C) Mmp9, (D) Acp5 and (E) Dc-stamp. (K) BMMs were incubated in an osteoclastogenic medium with vehicle or britanin for 3 days and the protein expression levels of Nfatc1 and Ctsk were determined using western blotting. BMMs were cultured with M-CSF (10 ng/ml) and RANKL (20 ng/ml) in the absence or presence of britanin for (F and G) 1 day and (H and I) 4 days, and RT-qPCR was performed to analyze the expression of (F) TRAF6, (G) TNF-α, (H) Nrf2, (I) Nqo1 and (J) HO-1. *P<0.05, **P<0.01 and ***P<0.001 vs. RANKL-treated control as analyzed by (A-E) ANOVA with Tukey's multiple comparison post hoc test and (F-J) two-tailed unpaired Student's t-test. Nrf2, nuclear factor erythroid-2-related factor 2; BMMs, bone marrow-derived macrophages; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor kB ligand; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Nfatc1, nuclear factor of activated T-cells, cytoplasmic 1; Ctsk, cathepsin K; Mmp9, matrix metallopeptidase 9; Acp5, tartrate-resistant acid phosphatase 5; Dc-stamp, dendritic cell-specific transmembrane protein; TRAF6, tumor necrosis factor receptor-associated factor 6; TNF-α, tumor necrosis factor-α; Nqo1, NAD(P)H quinone oxidoreductase 1; HO-1, heme oxygenase 1.