Fig. 3

RA B cells inhibit osteoblast differentiation and activate NF-κB and ERK signaling. a B cells were purified from 6-m-old WT and TNF-Tg mouse BM, as in Fig. 2A. Mesenchymal precursor cells (MPCs) were cultured for 2 days in OB-inducing medium + 20% CM from untreated (U) B cells or from cultures of previously stimulated B cells. These B cells had been treated for 24 h with 1/8, 1/4, 1/2, and 1/1 dilutions of the optimal concentrations of the stimulatory cocktail (2.5 μg/ml Anti-CD40+10 ng/ml IL4+10 μg/ml LPS). Culture plates were stained for ALP, and ALP+ areas were measured. *p < 0.05 vs. WT. b WT MPCs were cultured ± B cells in OB-inducing medium for 2 days. The area of ALP+ cells was measured. *p < 0.05 vs. WT. c Expression levels of Runx2 and ALP in cells from b were measured by qPCR. *p < 0.05 vs. WT. d B cells were isolated from BM or subchondral BM (SBM) of CIA mice and co-cultured with WT MPCs in OB-inducing medium for 2 days. The area of ALP+ cells was measured. *p < 0.05 vs. BM. e Expression levels of Runx2 and ALP in cells from d were measured by qPCR. *p < 0.05 vs. BM. f WT and TNF-Tg B cells were co-cultured with WT MPCs for 2 days. At the end of the culture period, B cells were removed and protein lysates from the MPCs were subjected to Western blot analyses. Expression of NF-κB, ERK, and β-catenin signaling molecules in cell lysates from MPCs were assessed. Supplementary Fig. 13 shows uncropped gel images. Each experiment was performed 3 to 8 times. Representative images and quantifications are shown from one independent experiment. All error bars represent s.e.m. Two-tailed unpaired Student’s t-test was performed