Figure 9

Functional interaction of STAT3 with NF-κB in SW 480 cells. A: NF-κB nuclear transfer in STAT3-decoy ODN-transfected SW 480 cells. Cells were transfected with STAT3-decoy ODN (ST3dODN) (2 μg/ml) (3 and 4), IL-6 (50 ng/ml, 16 h) (5 and 6), or mutated STAT3-decoy ODN (mtODN) (7 and 8). Controls: 1 and 2. B: Probe of STAT3-decoy ODN pull-down with NF-κB antibody in non-transfected cytoplasm and nuclear fractions (1 and 2) and in IL-6 (50 ng/ml)-treated cytoplasm and nuclear fractions (3 and 4). C: Analysis of NF-κB activity by pull-down assay with NF-κB-decoy ODN following treatment of cells with STAT3-decoy ODN (2 μg/ml). Cells treated with TNF-α (20 ng/ml) (3 and 4) transfected with STAT3-decoy ODN (2 μg/ml) (lanes 5 and 6), treated with both TNF-α and STAT3-decoy ODN (7 and 8), transfected with mutated STAT3-decoy ODN (2 μg/ml) (9 and 10), or with both TNF-α and mutated STAT3-decoy ODN (11 and 12); untreated cells: 1 and 2. Western blotting with NF-κB antibody (p50 or p65 subunits). D: mRNA levels of the NF-κB target I-κB (qPCR) after STAT3-decoy ODN treatment. Cells were not transfected, transfected with STAT3-decoy ODN (ST3dODN) (2 μg/ml), treated with TNF-α (20 ng/ml), treated with both STAT3-decoy ODN and TNF-α, or transfected with mutated STAT3-decoy ODN (mtdODN) (2 μg/ml) and TNF-α. E: mRNA levels of cyclin-D1 (qPCR) after treatment with STAT3-decoy ODN. Cells were not treated, treated with TNF-α (20 ng/ml), transfected with STAT3-decoy ODN (2 μg/ml), or treated with both TNF-α and STAT3-decoy ODN. Results are expressed as % of control.