Fig. 4

Escherichia coli lipopolysaccharides inhibits osteoblastogenesis and enhances osteoclastogenesis through NF-κB signaling. MSCs were treated with or without LPS, after culture, the cells were assayed for Alizarin red S staining (A). qPCR analysis for RUNX2 mRNA levels (B) and western blot analysis showing the expression of RUNX2, non-phosphorylated and phosphorylated (p) p65 (C). D ChIP assay for the interaction of p-smad1/5/8 with promoter of RUNX2 using anti-p-smad1/5/8 antibody or rabbit IgG. The input proteins served as controls. E Expression of RUNX2 in MSCs treated with or without LPS (100 ng/ml) or BAY11-7085 (NF-κB inhibitor) (10 μM). PreOCs were treated with or without LPS, after culture, the cells were assayed for TRAP staining (F), qPCR analysis of NFATc1 mRNA levels (G) and western blots showing the expression of NFATc1, non-phosphorylated and phosphorylated p65 (H). I ChIP assay for the interaction of p-p65 with promoter of NFATc1 using anti-p-p65 antibody or rabbit IgG. The input proteins served as controls. J Expression of NFATc1 in preOCs treated with or without LPS (100 ng/ml) or BAY11-7085 (10 μM). Data are averages ± SD. Each experiment was repeated three times. *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001. All P values were determined using one way ANOVA