Fig. 5

CircIKBKB directly interacts with recruits NF-κB to the promoters of multiple bone remodeling factors. a Real-time PCR analysis of mRNA levels of 25 bone-remodeling factor in the circIKBKB-overexpressing and control cells. GAPDH serve as a loading control. b ChIP assay analysis of the enrichment of p65 on the promoter of M-CSF and GM-CSF in the indicated cells. c ChIRP assay analysis of enrichment of circIKBKB on the promoter of M-CSF and GM-CSF in the p65-silenced and control cells. d Left and middle: Schematic illustration of the binding sites of circIKBKB and p65 on M-CSF promoter and PCR-amplified fragments of the M-CSF promoter. Right: ChIP and ChIRP assays analysis of enrichment of p65 and circIKBKB on the M-CSF promoter, respectively. e Left and middle: Schematic illustration of binding site of circIKBKB and p65 on GM-CSF promoter and PCR-amplified fragments of M-CSF promoter. Right: ChIP and ChIRP assays analysis of the enrichment of p65 and circIKBKB on the GM-CSF promoter, respectively. f Upper: Osteoclast differentiation assay by TRAP staining in the presence of CM from the indicated cells. Lower: Quantification of the number of TRAP⁺-multinuclear osteoclasts and TRAP activity from experiment in upper panel. g SEM images (left) and quantification of the number of resorption pits (right) of bone slices resorbed by pre-osteoclasts treated with CM from indicated cells. Each error bar represents the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001