Fig. 6

EphB4, TNFR2 and MAPK signaling pathways comprise a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation. a Levels of p38, p-p38, ERK1/2, p-ERK1/2, JNK1 + 2 + 3 and p-JNK1 + 2 + 3 in MC3T3-E1 cells treated with TNF-α for 0 min, 5 min, 15 min, 30 min and 60 min. b Levels of p38, p-p38, ERK1/2, p-ERK1/2, JNK1 + 2 + 3 and p-JNK1 + 2 + 3 in the pHBLV-TNFR2siRNA1 cells and the pHBLV-NC cells treated with or without 0.5 ng/ml TNF-α in regular culture medium for 15 min. c MC3T3-E1 cells were pretreated with or without 200 nM NVP-BHG712 in the regular culture medium for 1 h, and then 0.5 ng/ml TNF-α was added into the medium. The cells were incubated for another 15 min. Levels of ERK1/2 and p-ERK1/2 were determined. d-f MC3T3-E1 cells were cultured in the regular culture medium and pretreated with the ERK inhibitor U0126 (10 μM) for 1 h. The culture medium was then switched to the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α and U0126 (10 μM). Cells treated without U0126 (10 μM) served as controls. ALP activities were determined 7d or 14d after the treatment (d). mRNA levels (e) and protein levels (f) of BSP and RUNX2 were determined 3 days after the treatment. *, p < 0.05 vs. the control group; **, p < 0.01 vs. the control group