Figure 6.

JNK1 or JNK2 deficiency can both decrease IKKβ deficiency-sensitized osteoblast differentiation and mineralization. (A) ALP staining in Ikbkb^−/−, Ikbkb^−/− Mapk8^−/−, and Ikbkb^−/− Mapk9^−/− cells after 8 days of control medium or osteogenic medium incubation. (B) Microscopy images of Alizarin Red S staining of Ikbkb^−/−, Ikbkb^−/− Mapk8^−/−, and Ikbkb^−/− Mapk9^−/− cells induced by osteogenic medium for indicated days. (A,B) Results were representatives of three independent experiments. (C) Decreased mineralization in JNK1 or JNK2 deficiency cells as determined by de-staining Alizarin Red S and measuring absorbance at 560 nm (n = 3), compared with Ikbkb^−/− cells. Statistics by one-way ANOVA. (D) Western blot analysis for phospho-c-Jun in wildtype (WT), Ikbkb^−/−, Ikbkb^−/− Mapk8^−/−, and Ikbkb^−/− Mapk9^−/− cells after stimulated with β-GP and AA for indicated times points. (E,F) WT, Ikbkb^−/− knock out, Ikbkb^−/− Mapk8^−/−, and Ikbkb^−/− Mapk9^−/− double knock out cells were stimulated with β-GP and AA, and phosphorylation of c-Jun was analyzed by flow cytometry. (E) Histogram comparing c-Jun phosphorylation of WT, Ikbkb^−/−, Ikbkb^−/− Mapk8^−/−, and Ikbkb^−/− Mapk9^−/− cells 60 min after stimulation. (F) Mean fluorescence intensity (MFI) of phospho-c-Jun at 0, 60, and 120 min with β-GP and AA treatment (n = 3). Statistics by one-way ANOVA. (G) Real-time PCR results of mRNA levels of osteoblast differentiation marker genes, which showed significant decrease in JNK1 or JNK2 deficient cells (n = 3), compared with Ikbkb^−/− cells. Statistics by one-way ANOVA. Scale bars: 500 μm. Error bars represent ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.