Fig 2: 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.

Fig 3: Act1 (D10N) is a loss of function variant(a) Act1-/- MEFs transduced with vector, Act1 (WT), or Act1 (D10N) were subjected to immunoblot analysis for Act1 expression. (b) Act1-/- MEFs transduced as in (a) were treated with IL-17 (50ng/ml) for the indicated times. Cell lysates were subjected to gel-shift assay for NF-κB activation. (c) HEK293 cells transfected with E-selectin–luciferase reporter (100ng) and the indicated amounts of human Act1 or human Act1 (D10N) DNA, followed by luciferase assay analysis of NF-κB activity. (d) Act1-/- MEFs transduced as in (a) were treated with IL-17 (50ng/ml) for the indicated times, followed by immunoblot analysis. Arrow indicates Act1-modification. (e) Act1-/- MEFs transduced as in (a) were treated with IL-17 (50ng/ml) for the indicated times. Cell lysates were immunoprecipitated with anti-Act1, followed by immunoblot analysis for IL-17R, TRAF6, TRAF3, TRAF2, IKKi and Act1. Immunoprecipitated (IP) products are shown on left, whole cell lysates (WCL) are shown on right. (f) Act1-/- MEFs transduced with vector, Act1 (WT), Act1 (TB12), or Act1 (D10N) were left untreated or treated for 3 hours with TNF (10ng/ml), IL-17A (50ng/ml), IL-17F (50ng/ml), or TNF in combination with IL-17A, followed by RT-PCR analysis for Il6, Cxcl1 and Csf2 expression. The data are shown as fold induction over untreated (UT). *p < 0.05, **p < 0.01 and ***p < 0.005 (Student’s t-test). Data are representative of three independent experiments.

Fig 4: Effect of IKKi on the MAPK and AKT/GSK3ß/mTOR/FOXO/NF?B signaling pathways.(A–D) Levels of total and phosphorylated MEK1/2, ERK1/2, JNK, P38PI3K, AKT, GSK3ß, mTOR, FOXO and NF?B in the heart tissues of mice in the indicated groups (n = 6). A and C Representative blots. B and D Quantitative results. (E–F) Representative blots for total and phosphorylated AKT, GSK3ß, mTOR, FOXO3A, FOXO1 and NF?B after treatment with Ang II for the indicated times in H9c2 rat cardiomyocytes infected with Ad-GFP or Ad-IKKi. E Representative blots. F, Quantitative results. *P<0.05 vs. WT/sham; # P<0.05 vs. WT/AB after AB.

Fig 5: Effects of IKKi on cardiac hypertrophy.A, Echocardiographic results for the 4 groups of mice at 4 weeks following AB or sham surgery (n = 6). B, Statistical results of the HW/BW, LW/BW, and HW/TL ratios and myocyte cross-sectional areas of the indicated groups. C, Gross hearts, HE staining and WGA-FITC staining of the sham and AB mice at 4 weeks post-surgery. D, Expression levels of the transcripts of ANP, BNP, ß-MHC, a-MHC and SERCA2a after AB were determined by RT-PCR analysis (n = 6). E. Kaplan-Meier curve depicting the survival of WT-sham, KO-sham, WT-AB, and KO-AB mice *P<0.05 vs. WT/sham. # P<0.05 vs. WT/AB following AB.