Figure 4: NF-κB signaling is important for miR-155 expression in HTLV-1-positive T cells. (a) DNA-binding activities of proteins to the promoter region of miR-155 gene in HTLV-1-positive and -negative T-cell lines were evaluated by EMSA. The NF-κB oligonucleotide probe containing the NF-κB-binding site from miR-155 gene was used. Arrow shows specific DNA-protein complexes. (b) The specificity of NF-κB-DNA binding was analyzed in MT-2 cells. Competition assay with cold competitors of wild type (WT) or mutated (Mut) probe showed the specificity of the protein-DNA-binding complex. Antibodies to NF-κB subunits were used for super shift assay. The arrow shows specific DNA- NF-κB complexes. The supershifted complexes are indicated by an arrowhead. (c) An NF-κB inhibitor, Bay11-7082, inhibited miR-155 expression. Real-time RT-PCR shows miR-155 expression in MT-2 cells treated with different concentration of Bay11-7082. miR-155 expression is demonstrated as a fold induction relative to that in untreated cells. Values are the mean ± SD from three separate experiments. (d) The activity of NF-κB binding to the miR-155 gene promoter was suppressed by Bay11-7082. MT-2 cells were cultured with 0, 1, 5, or 10 μM of Bay11-7082 for indicated time periods. The NF-κB binding activity on miR-155 gene promoter was determined by EMSA. Specific DNA-NF-κB subunits complexes are indicated by the arrow.