Fig 2: Antiviral signal pathway was not involved in RANKL-induced activation of IKKe. (a) Cells were cultured in RANKL (50 ng/ml) and M-CSF for different time points. The expressions of Ifna and Ifnb were determined by qPCR (n=3). (b-c) WT, MAVS-/- or STING-/- cells were cultured in M-CSF for 3 days and then added RANKL (50 ng/ml) for indicated time point. The phosphorylation of IKKe induced by RANKL was evaluated by IB. (d) WT cells were cultured in M-CSF for 3 days and then added RANKL (50 ng/ml) together with IKKs inhibitor BAY-985 (2 nM) for indicated time point. The phosphorylation of IKKb and IKKe induced by RANKL was evaluated by IB. 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.

Fig 3: 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 4: 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 5: IKKe deficiency promoted osteoclastogenesis and expression of osteoclast-related genes. (a) TRAP staining was employed to analyze osteoclast differentiation from bone marrow cells. TRAP activity was determined by an ELISA reader at 410 nm (optical density). (b) Expression of osteoclast genes, including Cstk, Acp5 and Calcr, were determined by qPCR in the cells cultured for 3 days in RANKL (50 ng/ml) and M-CSF. (c) The levels of cytokines in WT and IKKe-/- osteoclast treated with LPS (100 ng/ml) were analyzed by qRT-PCR (n=4). 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.