Stat3 activation status was positively correlated with IL-6 expression and paclitaxel resistance in AS2-derived cells. (A) AS2-derived cells expressed various Stat3 mutant proteins. Cell lysates were collected 24 hours after seeding. The total amount of Stat3 protein and Stat3 phosphorylation level (pStat3-Y705) were analyzed by Western blot analysis. (B) Stat3 activation status positively correlated with IL-6 mRNA expression. RNA samples were collected 24 hours after seeding. The expression of IL-6 mRNA was analyzed by RT-PCR. IL-6 mRNA levels of each cell were normalized to β-actin mRNA levels and indicated as a relative fold to that of the parental AS2 cell. (C) Stat3 positively regulated IL-6 promoter luciferase activity. The p1168huIL6P-luc+ IL-6 reporter plasmid and the control phRL-TK plasmid were co-transfected without (mock) or with control vector (Vec) or S3C plasmid or S3D plasmid into AS2 cells. The cells were incubated for 24 hours and then lysed and subjected to Firefly and Renilla luciferase activity measurements. This graph shows the results as mean ± SEM. Student's t tests, ***p < 0.001. (D and E) Stat3 activation status positively correlated with IL-6 secretion. The cells were seeded for 24 hours before medium replacement and the culture supernatants were collected 1, 3, 8, or 24 hours after medium replacement. IL-6 secretion was analyzed by ELISA. The graphs show the results as mean ± SEM. Student's t tests, **p < 0.01 and ***p < 0.001. For a clearer demonstration, statistical significances are shown for the 24-hour time points only. (F) Stat3 activation status positively correlated with paclitaxel resistance. Cells were seeded for 24 hours and then treated with paclitaxel at the indicated doses for another 72 hours. Cell viability was determined by MTT assay. The graph represents the results as mean ± SEM. Student's t tests, *p < 0.05, ## p < 0.01 and ***p < 0.001 (For a clearer demonstration, the p of AS2/S3F-3 were shown as #.).