Inhibition of STAT1 activation by siRNA. By Western blot analysis, the protein level of STAT1 and phospho-STAT1 were dramatically decreased in KYSE150 and KYSE510 treated with siRNA against STAT1. Cell lysates were collected 2 days after the siRNA transfection (A). The decrease in STAT1 expression after siRNA treatment was further supported by quantitative RT-PCR (***p < 0.0001) (B). In both KYSE150 and KYSE510, siRNA knockdown of STAT1 induced a significant decrease in cell growth, assessed by trypan blue eclusion assay. The cell numbers were assessed on day 4 after siRNA transfection. Triplicate experiments were performed and the results of a representative experiment are illustrated (*p < 0.05) (C). Transfection of STAT1 siRNA into KYSE150 and KYSE510 cells led to a significant reduction in the number of colonies formed, as compared to cells transfected with scrambled siRNA. Triplicate experiments were performed and the results of a representative experiment are shown (***p < 0.0001) (D). By western blots, transfection of STAT1 siRNA resulted in an appreciable increase in BCL-xL, BCL-2, cyclin D1 and a corresponding decrease in p21waf1. Cells treated with scrambled siRNA served as the negative controls. Cell lysates were prepared two days after siRNA transfection (E). Cell cycle analysis using flow cytometry revealed that STAT1 siRNA induced a significant decrease in the sub-G1 fraction in both cell lines, KYSE150 and KYSE510 (*p < 0.05). Results shown are representative of three independent experiments (F).