Fig. 6

EIF4A3 increases circIKBKB expression via direct promotion of circIKBKB cyclization. a Schematic illustration of circIKBKB pre-mRNA pull-down following the mass spectrometry to identify circIKBKB cyclization factor. b Real-time PCR analysis of circIKBKB expression in the indicated SCP2 cells. GAPDH served as a loading control. c Real-time PCR analysis of circIKBKB and IKBKB expression in EIF4A3-overexpressing and control MDA-MB-231 cells. GAPDH served as a loading control. d The putative binding sites of EIF4A3 in the upstream and downstream region of the circIKBKB pre-mRNA predicted with circinteractome database (left), and RIP assay analysis of interaction of EIF4A3 with circIKBKB pre-mRNA in SCP2 cells. The IKBKB intron 1 was used as the negative control and H19 lncRNA was used as the positive control. e A schematic diagram of 7 fragments of circIKBKB pre-mRNA (upper) and RNA pull-down assay analysis (Lower) of the interaction between eIF4A3 and above 7 fragments of circIKBKB pre-mRNA. f Relative NF-κB-driven luciferase activity was analyzed in the indicated cells treated with TNF-α (2 ng/ml). g WB analysis of the 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. h ELISA analysis of expression of secreted M-CSF and GM-CSF in CM from the indicated cells. i Image (left) and quantification (right) of TRAP⁺-multinuclear osteoclasts stimulated by CM from the indicated cells. Each error bar represents the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001