Figure 2.

Enhanced osteogenic differentiation and matrix mineralization in IKKβ-deficient MC3T3–E1 cells. (A) Phosphorylation of p65 was examined by Western blot analysis in steady state. The phosphorylation of p65 at Ser536, an important site of IKKβ action was significantly inhibited. (B) The wildtype (WT) control and Ikbkb^−/− clone 19, 24, 26 were cultured in the control medium or osteogenic medium for 8 days. ALP activity was analyzed using ALP staining. (C) Microscopy images of Alizarin Red S staining of WT control and Ikbkb^−/− cells induced by osteogenic medium. (B,C) Results were representatives of three independent experiments. (D) Mineralization described in (C) was quantified by de-staining Alizarin Red S with 10% cetylpyridinium chloride and the absorbance was measured at 560 nm (n = 3), the level of mineralization in Ikbkb^−/− cells was compared with WT cells. Statistics by one-way ANOVA. (E) FACS plots showing non-transfected control and cells transfected with Ikbkb targeting plasmids. We sorted 10,000 DsRed2/ECFP double-positive cells as Ikbkb^−/− cells bulk, and subjected to phenotype experiment. Alizarin Red S staining (F) and quantification (G) of Ikbkb^−/− cells bulk induced by osteogenic medium for 16 days compared with WT cells (n = 3). Statistics by two-tailed t-test. (H) The deletion of IKKβ increased mRNA levels of osteoblast differentiation marker genes as determined by real-time polymerase chain reaction (n = 3), compared between WT and IKKβ-deficient cells. Statistics by one-way ANOVA. Scale bars: 500μm. Error bars represent ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.