Knocking-down Stat3 by stable transfection with shRNA decreased the expression of IL-6 in AS2 cells. (A) Stable cell lines were transfected with plasmid containing Stat3 shRNA (AS2/shStat3-1 and AS2/shStat3-2) or control plasmid (AS2/shVec) and selected with 300 μM Hygromycin-B. The 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) The mRNA expression of IL-6 was decreased in stable cell lines expressing Stat3 shRNA. The cells were seeded 24 hours before medium replacement. RNA samples were collected 0, 1 and 3 hours after medium replacement and IL-6 mRNA expression was measured by RT-PCR. (C) The secretion of IL-6 was decreased in stable cell lines expressing Stat3 shRNA. The cells were seeded 24 hours before medium replacement. Culture supernatants were collected 1, 3, 8, or 24 hours after medium replacement. IL-6 secretion was measured by ELISA. The graph represents the results as mean ± SEM. Student's t tests, ***p < 0.001. For a clearer demonstration, statistical significances are shown for the 24-hour time points only. (D) Stat3 shRNA expressing cells showed lower resistance to paclitaxel and pretreatment with exogenous IL-6 modestly restored the resistance. Cells were seeded for 24 hours and then treated as following: (1) cells without pretreatment and treated with varying doses of the chemotherapeutic agent paclitaxel only for 72 hours, and (2) cells pretreated with exogenous IL-6 (20 ng/ml) for 1 hour and then cotreated with exogenous IL-6 + paclitaxel for 72 hours. The viability of cells was analyzed by MTT assay. The graph represents the results as mean ± SEM. Student's t tests, ***p < 0.001 and ###p < 0.001 (For a clearer demonstration, the p of AS2/shStat3-2 were shown as #.). For a clear demonstration, statistical significances between the IL-6 pre-treated and un-pretreated groups were not shown.