Figure 2
sh/siRNA-mediated knockdown of ILK suppresses endogenous and IL-6-stimulated upregulation of MUC1-C expression without altering MUC1 mRNA expression. (a) Effect of shRNA-mediated silencing of ILK versus control on the phosphorylation and/or expression of STAT3 and MUC1-C in IL-6-treated AsPC-1 or AsPC-1IL-6 cells. For ILK depletion, AsPC-1 cells were transfected with ILK shRNA using lentivirus infection to generate a stable knockdown clone. For exogenously added IL-6, cells were treated with IL-6 at the indicated concentrations for 24 h. (b) shRNA-mediated knockdown of E2F1 suppressed the expression of ILK and MUC1-C in AsPC-1 cells. (c) The suppressive effect of ILK depletion on endogenous and IL-6-induced upregulation of MUC1-C expression is independent of STAT3. ILK silencing did not affect the cellular distribution and phosphorylation status of STAT3 in AsPC-1 cells. The stable ILK-depleted cells were treated with vehicle or IL-6 (50 ng/ml) for 24 h. (d) qRT-PCR analysis of the effect of siRNA-mediated ILK depletion versus control on MUC1 mRNA expression in IL-6- versus vehicle-treated AsPC-1 cells (means±s.e., n=3). Scrb, scrambled (control) siRNA. Cells were transiently transfected with ILK siRNA for 48 h followed by treatment with IL-6 (50 ng/ml) for an additional 24 h, and subjected to qRT-PCR analysis. (e) Left, effect of siRNA-mediated knockdown of ILK on the expression of endogenous MUC1-C in AsPC-1 and SW1990 cells. Cells were transfected with ILK siRNA for 72 h. Right, qRT-PCR analysis of the effect of ILK depletion versus control on MUC1 mRNA expression in AsPC-1 and SW1990 cells (means±s.e., n=5 and 3, respectively). Cells underwent the same treatment before qRT-PCR analysis for MUC1 mRNA expression. In western blot analysis, data shown are representative of at least three independent experiments.
