Fig. 4

Regulation of NF-κB during osteogenic differentiation and impact of NF-κB inhibition on mineralization. a, b Expression of NF-κB (p65 subunit, a) and P-NF-κB (p65 subunit, Ser536, b) in DFCs after cultivation in control medium (DMEM), osteogenic differentiation medium (ODM) or BMP2 containing differentiation medium for 1, 7, 14 or 28 days was determined by Western blot analysis. The phospho-ratio (b) was calculated as ratio between phosphorylated and total NF-κB. c, d DNA binding activity of NF-κB subunits p50 (c) or p65 (d) in DFCs after 7 days cultivation in control or differentiation media as above, measured by NF-κB activity assay. e DFCs were cultivated in the control medium (DMEM) or osteogenic differentiation medium (ODM) and simultaneously treated with either 200 nM PKC activator PMA alone or in combination with 500 nM NF-κB inhibitor CID2858522 for 28 days. Mineralization was determined by Alizarin Red staining. Bar charts show means + SD (n = 3). One-way ANOVA was performed to compare different media at the same time point including Tukey’s post hoc tests comparing the individual differentiation media with the control medium (a–d) or to compare all groups including Tukey’s post hoc tests comparing the different groups in ODM pairwise or DMEM to ODM control group (e). *p < 0.05, **p < 0.01, ***p < 0.001