Fig 2: IKKe presented protective effects in bone tissues in methylprednisolone-treated mice. (a) Sagittal images of the bones within the femoral heads in MPS-treated WT and IKKe-/- mice. (b) H&E images of the femoral heads in WT and IKKe-/- mice treated with MPS. Scale bar: 100 μm. (c) mRNA levels in bone tissue from WT and IKKe-/- mice with ONFH were examined using qRT-PCR (n=4). All data are presented as folds were calculated based on the Actin by Bio-Rad software. Data are mean ± SEM. Representative experiments were performed at least three independent times. *P < 0.05.

Fig 3: The expression of IKKe is reduced in ONFH patients. (a) The CD11b+ cells in PBMCs from healthy donors and ONFH patients were isolated by FACS. The differences of multiple gene expressions in were analyzed by RNAseq. (b-c) The mRNA (b) and protein level of IKKe in CD11b+ cells in PBMCs from healthy donors and ONFH patients were measured by qPCR (n=4) and immunoblot (n=3) (c). (d) Cells were cultured in RANKL (50 ng/ml) and M-CSF for indicated days. The phosphorylation of IKKe induced by RANKL was evaluated by IB at indicated time point. Relative mRNA folds for each gene were calculated based on the Actin by Bio-Rad software. Data are mean ± SEM. Representative experiments were performed at least three independent times. *P < 0.05.

Fig 4: IKKe negatively regulate the protein level of NIK. (a) The interacted proteins of NIK were presented in STRING Interaction Network. (b) HEK293 cells were transfected with various combinations of expression vectors for 48 h, then the whole cell lysates were subjected to Western blot analysis. (c) Immunoassay of HEK293T cells transfected with various combinations of expression vectors for HA-tagged IKKe (HA-IKKe) and Myc-tagged NIK (HA-NIK) followed by immunoprecipitation of proteins from lysates. (d) Immunoblot analysis of NIK and actin in whole-cell lysates of wild-type and IKKe KO osteoclasts stimulated with RANKL (50 ng/ml). Data are mean ± SEM and representative of at least three independent experiments.

Fig 5: MRTFs suppress TBK1 phosphorylation in bronchial and coronary SMCs. Human bronchial SMCs were transduced with null or MRTF-A virus for 8 days. Cells were harvested, and proteins were isolated for western blotting (a). We assayed phosphorylation of the kinases TBK1 (p-TBK1, Ser172) and IKKε (p-IKKε), three contractile differentiation markers (markers), and four loading controls (loading ctrls) in the same lysates. Panel (b) shows analysis of p-TBK1 and total TBK1 versus proteins remaining on the gel (n = 6, unpaired t-tests), p-TBK1 versus t-TBK1, and p-TBK1 versus H3. Panel (c) shows levels of TBK1 and IKBKE in spleen (n = 241), tibial artery (n = 663), aorta (n = 432), coronary artery (n = 240), and lung (n = 578) (in transcripts per million, TPM, from GTExPortal.org). The ratio of median expression (TBK1/IKBKE) was calculated for all tissues. This ratio was considerably higher in arteries and lung than in spleen. In new and independent experiments, human bronchial SMCs were next transduced with either null or MRTF-A virus and phosphorylation of TBK1 (Ser172) versus total TBK1 was assessed using western blotting (d). Summarized data after normalization of p-TBK1 to total TBK1 is shown in (e) (n = 6, unpaired t-test). Panels (f) and (g) show similar analyses using coronary artery SMCs (n = 6, Mann Whitney test). In (h) and (i), coronary SMCs were transduced in parallel with null, MRTF-A, MRTF-B, and MYOCD viruses followed by western blotting for p-TBK1/TBK1 (n = 12, Kruskal Wallis ANOVA, with Dunn’s post-hoc test). *p < 0.05, **p < 0.01, ****p < 0.0001.