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Figure 5 | Genome Biology

Figure 5

From: NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation

Figure 5

NF-κB dependence of miRNA expression changes. (A) Analysis of the presence of NF-κB subunit binding motifs (from TRANSFAC database) in a 1,000-bp window centered around the estimated TSS of the miRNAs. (B) ChIP assays for selected miRNAs showing the binding of NF-κB p65 near the TSS 2 and 4 days after RANKL/M-CSF stimulation of MOs. Each graph contains the relative enrichment of samples immunoprecipitated with the anti-p65 antibody and an IgG as a control. MO samples were tested at 0, 2 and 4 days after M-CSF/RANKL stimulation. On top of each graph the sequence analyzed is indicated. p65 putative binding sites are indicated with a blue dot. Primers used for amplification are indicated with arrows around p65 binding sites. (C) Effects of the two NF-κB inhibitors (10 μM BAY 11-7082 (BAY11) and 100 μM sodium aurothiomalate (SATM) on the phosphorylation levels of p65 as determined by western blotting. p65 and H3 total levels are used as controls. (D) Effects of two NF-κB inhibitors (BAY11 and SATM) on the levels of miRNAs within the miR-99b/125a/let7e and miR-132/212 clusters measured by a time-course analysis of MOs stimulated with RANKL/M-CSF. (E) Effects of treatment with BAY11 and SATM on markers of OC differentiation (ACP5, CTSK, TM7SF4, MMP9) as estimated by qRT-PCR. Data relative to DMSO-treated samples and normalized with RPL38 expression levels. (F) Effects of treatment with BAY 11 and SATM on miRNA targets (IGF1R, TNFAIP3, ITGA4, THBS, IL15 and PTGS2) as estimated by qRT-PCR. Data are relative to DMSO-treated samples and are normalized with respect 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.

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