Figure 7.

TNF induces STAT3 nuclear translocation and DNA binding. HCEC cells were treated with 0.66 ng/mL TNF for 24, 48, or 72 hours. A: Cytoplasmic and nuclear proteins were separated to analyze STAT3 distribution by immunoblotting with anti-pSTAT3^Y705, anti-STAT3, and anti-histone H1/anti-GAPDH antibodies. Western blots are representative of three independent experiments. B: DNA binding activity of STAT3 in nuclear extracts was evaluated by nonradioactive EMSA using infrared labeled oligonucleotides containing the STAT3 consensus sequence in the presence or absence of 100× competitor (Com) or mutant (Mut) oligos. Results are representative of two independent experiments. C: Theoretical image of the DAPK promoter showing STAT3 binding motifs distributed in two regions. D and E: Normal human colon epithelial cells were treated with 0.66 ng/mL TNF for 48 hours. The ChIP assay was performed by using IgG or anti-pSTAT3^Y705 antibodies. End-point PCR followed by agarose gel electrophoresis (D) and quantitative PCR (E) were used to analyze STAT3 binding to both regions of the DAPK promoter. Data are derived from four independent experiments performed in duplicate. *P < 0.05 versus the corresponding control, U-test.