Fig. 3

Activation of NF-κB is essential for circIKBKB-induced BC bone-metastasis. a Cignal finder signal transduction 45-pathway reporter array showing that circIKBKB overexpression significantly activated NF-κB signaling in BC cells. b Relative NF-κB-driven luciferase activity was analyzed in the indicated cells treated with TNF-α (2 ng/ml). c EMSA of the endogenous NF-κB activity in the indicated cells. Oct-1/DNA-binding complex was used as a control. d Subcellular localization of NF-κB p65 in the indicated cells as analyzed by an immunofluorescence staining assay. e Western blotting analysis of level of cytoplasmic-p65, nuclear-p65, total p65, p-IKK-β, total IKK-β, p-IκB-α, and total IκB-α in the indicated cells treated with TNF-α (2 ng/ml). GAPDH served as a cytoplasmic control and p84 served as a nuclear control. f Western blotting analysis of the K48-linked polyubiquitin levels of IκB-α in the indicated cells. g Upper: Osteoclast differentiation assay by TRAP staining in the presence of CM from vehicle and IKK2-I VI-treated cells. Lower: Quantification of the number of TRAP⁺-multinuclear osteoclasts and TRAP activity from experiment in the upper panel. h SEM images (upper) and quantification of the number of resorption pits (lower) of bone slice resorbed by pre-osteoclasts in the presence of CM from vehicle and IKK2-I VI-treated cells. i BLI, μCT (longitudinal and trabecular section) and histological (H&E and TRAP staining) images of bone lesions from vehicle- and IKK2-I VI-treated mice. Scale bar, 50 μm. Right: Quantification of μCT osteolytic lesion area and TRAP⁺ osteoclasts along the bone-tumor interface of metastases from experiment in the left panel. Each error bar represents the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001