Figure 1.

TNF and TNF+RANKL induce OC formation from TRAF6^−/− OCPs cultured on plastic. A, representative sections of tibiae from 12-day-old WT (+/+) and TRAF6 KO (−/−) mice stained for TRAP activity (red staining). OC numbers (Oc.N) in the tibial secondary ossification centers and metaphyses were counted. **, p < 0.01 for −/− mice (n = 7) versus +/+ mice (n = 5). B, 2 × 10⁵ spleen cells from 12-day-old +/+ and −/− mice were treated with M-CSF for 2 days to recruit OCPs, followed by treatment with TNF (T, 20 ng/ml) or RANKL (R, 10 ng/ml) for 3 days in 96-well plates. TRAP+ OC numbers and areas (Oc.Ar) were counted. **, p < 0.01 versus the respective RANKL-treated cells; ##, p < 0.01 versus the respective TNF-treated cells; n = 4/group. C, OCPs were cultured with TNF and M-CSF for 10 days on bone slices in 96-well plates. The bone slices were fixed with 10% formalin, and the cells on them were brushed off followed by 0.1% toluidine blue staining. Resorption pit areas were counted. All in vitro experiments were repeated at least twice with similar results.