Figure 6.

TRAF3 limits OC differentiation in the absence of TRAF6. A, BM cells from 2-month-old TRAF3^f/f-LysM^cre (TRAF3 conditional knock out in myeloid cells, −/−) mice and their WT (+/+) littermates were cultured with RANKL (R), TNF (T), TGFβ1 (Tβ1), or combinations of them in the presence of M-CSF for 5 days. TRAP staining was performed to evaluate OC numbers (Oc.N) and area (Oc.Ar). *, p < 0.05; **, p < 0.01 between cKO and WT cells. Western blotting was performed on cell lysates from PBS- and TNF-treated WT and TRAF3^f/f-LysM^cre OCPs and shows the absence of TRAF3 in the cells from TRAF3^f/f-LysM^cre (bottom right panel). B, 2 × 10⁵ WT or TRAF6^−/− spleen cells were cultured in 96-well plates with M-CSF for 2 days and then infected with GFP or TRAF3 retroviral supernatant. 1 day after infection, cytokines were added to the cultures for 3 days to generate OCs. OC numbers were counted after TRAP staining (top right panel). *, p < 0.05 versus its respective level in GFP-infected cells. TRAF3 protein levels were assessed in OCPs infected with GFP or TRAF3 retroviruses by Western blotting (bottom right panel) to confirm overexpression of TRAF3. All experiments were repeated at least twice with similar results. T, TNF, 20 ng/ml; R, RANKL, 10 ng/ml; Tβ1, TGFβ1, 1 ng/ml.