Stat3 contributed to the elevation of IL-6 in drug resistant cancer cells. (A and B) KB-CPT100 and MCF-7/ADR cells exhibited higher drug resistance than their parental KB and MCF-7 cells. The cells were seeded for 24 hours and treated with camptothecin (KB-CPT100 and KB cells) or epirubicin (MCF-7/ADR and MCF-7 cells) at the indicated doses for another 72 hours. The viability of cells was determined by MTT assay. (C and D) IL-6 expression was elevated in the two drug resistant cancer cells: KB-CPT100 and MCF-7/ADR compared to their parental cells. 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 determined by ELISA. The graphs (A-D) represent the results as mean ± SEM. Student's t tests, **p < 0.01 and ***p < 0.001. (E and F) Transient transfection with Stat3 siRNA effectively knocked-down Stat3 in these two drug resistant cells. Cells were left untreated as controls (C), transfected with nothing as mocks (M), or transfected with 50 nM of scramble control siRNA or Stat3 siRNA (Stat3#1). The cells were incubated for 72 hours and then cell lysates were collected. The total amount of Stat3 protein and Stat3 phosphorylation level (pStat3-Y705) were analyzed by Western blot analysis. (G and H) IL-6 secretion was suppressed by transient transfection with Stat3 siRNA in these two drug resistant cells. Medium replacement was performed 72 hours after transfection. Culture supernatants were collected 3, 8, or 24 hours after medium replacement and IL-6 secretion was determined by ELISA. The two graphs represent 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.