Figure 3

Camptothecin blocks STAT3 activation and the interaction of STAT3 and the gp130 receptor. (A) Western blot analysis for the indicated proteins from HCT116 cells transfected with STAT3 cDNA and then treated with 250 nM CPT for 16 h. (B) HCT116 cells were transfected with an IRF-1 reporter plasmid to measure STAT3 activation along with JAK1 and 2 cDNAs. After 48 h, cells were washed and treated 250 nM CPT. After 24 h, the samples were harvested and washed twice before being lysed and combined with a luciferase assay reporter. The data is reported as the mean +/- s.d. of 2 independent experiments performed in triplicate. (C) HCT cells were transfected with STAT3 cDNA and gp130 cDNA. After 48 h, Samples were treated with 40 ng/ml IL-6 or IL-6 and 250 nM CPT. Samples were divided and either saved for Western blot analysis (input) or incubated with an antibody to gp130 for 6 h. Protein G agarose beads were added and the samples rotated over night. Western blot analysis was performed using the IP supernatant and examined for the indicated proteins. In comparison to empty vector controls (EV), the relative activity of STAT3 transcription was increased by: *JAK1, p < 0.0005; **JAK2, p < 0.0001. In the presence of CPT, JAK1-mediated STAT3 transcription was inhibited #JAK1 + CPT p < 0.0002 and JAK2 inhibited ## JAK2 + CPT p < 0.0003. The data represents the mean +/- s.d. of 2 independent experiments performed in duplicate.