Figure 4

The inhibitory effect of CM-Adipo on BMP2-induced osteogenesis is mediated by activating NF-κB signaling pathway. b Western blot analysis of NF-κB subunit p-65 phosphorylation in mBMSCs cultured in CM-Adipo versus CM-Control. Cells were incubated with the CM for 30 min and cell laystes were subjected to western blot analysis. b CM-Adipo stimulates NF-κB signaling activity. HEK 293 cells were transfected with Cignal NF-κB Reporter negative control, or positive control. Cells were incubated with 50% and 100% CM-Control or CM-Adipo for 24 h. Dual-luciferase assays were performed, and reporter activity was represented as arbitrary units after normalization to the internal Renilla reporter. c Effect of NF-κb inhibitor, BAY 11-7082 on retrieving the inhibition of BMP2-induced osteoblast differentiation, as measured by ALP activity quantification and (d) Alizarin Red staining of matrix mineralization. Cells were treated with different concentrations of the inhibitor, 1 h prior the treatment with BMP2. ALP activity was measured after 6 days and represented as fold change after normalization to the cell viability. Alizarin Red was measured after 10 days. Values are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005)