Knocking-down Stat3 by transient transfection with synthetic siRNA decreased IL-6 expression in AS2 cells. (A) Transient transfection with synthetic Stat3 siRNA effectively knocked-down Stat3 expression. AS2 cells were left untreated as controls (C), transfected with nothing as mocks (M), or transfected with two different doses 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. (B) Transient transfection with Stat3 siRNA decreased IL-6 mRNA expression. 72 hours after transfection, the medium was replaced and RNA samples were collected 24 hours afterwards. IL-6 mRNA expression was measured by RT-PCR. IL-6 mRNA levels of each sample was normalized to β-actin mRNA levels and indicated as a relative fold to that of the control. (C) Transient transfection with Stat3 siRNA decreased IL-6 secretion. 72 hours after transfection, the medium was replaced and culture supernatants were collected 3, 8 and 24 hours afterwards. IL-6 secretion was measured by ELISA. The graph represents 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. (D) Knocked-down Stat3 did not affect cell viability. The viability of cells was determined by MTT assay 72 hours after transfection. The graph represents the results as mean ± SEM.