Figure 3.

Deletion of IKKβ increases phosphorylation of JNK in both MC3T3–E1 cells and primary calvarial osteoblasts. (A,B) Flow cytometry analysis of phospho-JNK in MC3T3–E1 wildtype (WT) cells and IKKβ-deficient cells after β-GP and AA stimulation. (A) Representative data of phosphorylation of JNK at 30 min after β-GP and AA stimulation. Fluorescence intensity for phospho-JNK was compared using histogram overlay between fluorescence minus one (FMO) control (gray), WT cells (Blue), and IKKβ-deficient cells (Orange). (B) Mean fluorescence intensity (MFI) of phospho-JNK at 0, 15, 30, and 60 min after β-GP and AA treatment, compared between WT and IKKβ-deficient cells (n = 6). Statistics by one-way ANOVA. (C) Western blot analysis for phospho-JNK in WT and IKKβ-deficient MC3T3–E1 cells after stimulating by β-GP and AA for indicated time points. Results were representatives of three independent experiments. (D) FACS plots showing primary osteoblasts transfected with pX458 empty backbone plasmid (left panel) as control and pX458-Ikbkb-targeting plasmids (right panel). The plasmids could express EGFP reporter. By gating on the EGFP positive primary osteoblasts, we distinguished the two groups of cells which were successfully transfected with empty backbone plasmid or IKKβ targeting plasmids, and compared their MFI of phospho-JNK at 0, 30 min after β-GP and AA stimulation (E,F). (E) Showing representative data at 30 min after β-GP and AA treatment. (F) Differences of phospho-JNK MFI between Ikbkb targeting primary osteoblasts and backbone plasmid transfected cells, with or without β-GP and AA treatment (n = 3). Statistics by two-tailed t-test. (G) Representative immunofluorescence images of phospho-JNK (green) and nuclei (blue) staining in WT and IKKβ-deficient MC3T3–E1 cells at 0, 30 min after the stimulation by β-GP and AA. Scale bars: 50 μm. Error bars represent ± SEM. *P < 0.05, ***P < 0.001, ns = not significant.