Fig 2: JNK1 or JNK2 deficiency can both decrease IKKß 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 ß-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 ß-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 ß-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.

Fig 3: The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of IRF3 and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).

Fig 4: Increased death sensitization of Ikk2-deficient SFs in TNF-mediated signals. a Representative images depicting TUNEL+ or CC3+ cells in sections of Ikk2f/f and Ikk2Ms-KO mice (naive, hTNFtg (week8) or CAIA-treated (D8)) (n = 6 per genotype). Note the CC3+ cartilage cells (blue arrow), lining (black) and sublining (red) SFs, and other cells (open arrow) within necroptic areas. b Quantitation of TUNEL+ cells found in synovial membrane area of Ikk2f/f and Ikk2Ms-KO in naive (n = 5–7) and arthritic conditions (hTNFtg (n = 4–7); CAIA (n = 4)). c Survival rates of Ikk2f/f, hTNFtg Ikk2f/f SFs, and WT MEFs treated with different TNF concentrations, in the presence or absence of ML120b inhibitor (representative experiment, n = 5). d Representative immunoblot of IKK2 expression after the application of TAT-Cre in SF cultures derived from Ikk2f/f and hTNFtg Ikk2f/f mice, and IkB degradation and JNK1/2 phosphorylation patterns after TNF or IL-1β treatment of Ikk2-deficient SFs. Relative quantitations are depicted for each blot. e Representative survival rates of Ikk2Δ/Δ and hTNFtg Ikk2Δ/Δ SFs and Ikk2−/− MEFs and control Ikk2f/f and hTNFtg Ikk2f/f SFs and WT MEFs treated with different TNF concentrations (n = 15, 4 experiments). Scale bar: 100 μm. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by two-tailed Student’s t-test (b) or one-way ANOVA (c, d). Unprocessed original scans of blots are shown in Supplementary Fig. 8

Fig 5: Enhanced osteogenic differentiation and matrix mineralization in IKKß-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 IKKß 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 IKKß increased mRNA levels of osteoblast differentiation marker genes as determined by real-time polymerase chain reaction (n = 3), compared between WT and IKKß-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.