Fig. 1.

Lipopolysaccharide (LPS) and tumor necrosis factor-a (TNF-α) suppress MFG-E8 expression in vitro. Bone marrow stromal cells were differentiated for 7 days into osteoblasts and treated with LPS (1 mg/mL) or TNF-α (50 ng/mL) for 48 hours. Vehicle-treated cells were used as controls (CO). (A) Gene expression of MFG-E8 was determined using qPCR and normalized to β-actin. (B) Protein levels were determined using Western blot analysis. One representative blot is shown. Numbers indicate mean ± standard deviation of 4 independent experiments. (C) Cells were isolated from wild-type (WT) or Toll-like receptor 2/4 knock-out (TLR2/4 KO) mice and stimulated with LPS (1 μg/mL, 48 hours). Gene expression of MFG-E8 was determined using qPCR and was normalized to β-actin. (D) Cells were pretreated with sTNFR1 (1 μg/mL) or sTNFR2 (1 μg/mL) for 1 hour before cells were treated with 50 ng/mL TNF-α for 48 hours. MFG-E8 expression was determined using qPCR. n = 4–6 mice per experiment. *p < 0.05; **p < 0.01.