Figure 2

Camptothecin (CPT) reduces phosphorylation of STAT3 and RKIP induced by interleukin-6 (IL-6). (A) Western blot analysis of: a dose-dependent reduction of STAT3 phosphorylation after IL-6 treatment then treated with 250–750 nM CPT; HCT116 cells treated with 250 nM CPT and 40 ng/ml IL-6 for 12 h. (B) The same co-treatment experiment as in (A) was repeated with HCT116 cells treated with 250 nM CPT and 40 ng/ml IL-6 for 12 h. Western blot analysis was performed to examine the protein levels of pRKIP, RKIP and actin. (C) Cell extracts were prepared after 18 h following treatment with CPT, IL-6 or the combination for flow cytometric analysis to examine binding to 7-AAD and annexin-V (% Apoptosis). The % Apoptosis of each sample is indicated in the top right corner of every panel: a) untreated control cells (0.48%); b) CPT (17.68%); c) IL-6 (0.64%); d) CPT + IL-6 (10.94%). The figure is representative of part of 1 experiment performed in duplicate. The experiment was repeated twice. (D) HCT116 cells were transfected with an IRF-1 reporter plasmid for STAT3 activation. After 48 h, the cells were washed and treated with 40 ng/ml IL-6, 250 nM CPT, or the combination. After 24 h, 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. A paired t-test was performed to analyze the increase in STAT3 transcription of IL- 6 treated experimental samples when compared to vehicle (CTR): *IL-6, p < 0.000012; or decrease when comparing IL-6 to samples treated with **IL-6 and CPT, p < 0.0002.