Figure 6

NF-κB p65 has a direct role in changes in miRNA expression levels. (A) Diagram depicting the region of the p65 gene in exon 11 targeted by the siRNA used in this study. Effects of siRNA experiments on p65 levels in MOs stimulated with RANKL/M-CSF after 1, 2, and 4 days, as analyzed by western blotting (bottom and central panels, normalized with respect to histone H3 levels) and qRT-PCR (left panel, relative to RPL38 expression levels). (B) Effect of p65 depletion on its recruitment near the TSS of the coding sequence of the miRNAs, as demonstrated by ChIP assays. The scheme on top of each graph depicts the region analyzed, indicating the p65 binding site (dot) and the primers used (arrows around the p65 binding site). (C) Effects of p65 siRNA experiments on miR-99b, miR-125a and miR-let7e after 1, 2, and 4 days. Data relative to miR-103 levels. (D) Effects of p65 downregulation on expression of genes upregulated during osteoclastogenesis (CTSK, MMP9, ACP5, TM7SF4). (E) Effects of p65 downregulation on the levels of the miRNA targets TNFAIP3 and IGF1R. Expression data compared with MO samples treated with control siRNA and values relative to RPL38 expression levels. Error bars correspond to the standard deviation of three independent measurements; *corresponds to P-value <0.05; **means P-value <0.01.