Avicin D downregulates Stat3-induced protein expression.

(A) Effect of avicin D on Stat3 -mediated luciferase gene expression. U266 cells were transfected with Ly6E-Luciferase-Stat3 plasmid as described in the methods. 24 hrs later cells were washed and treated first with avicin D (1 µM) for 16 hrs, and then exposed to IL-6 (10 ng/ml) for 30 min. Luciferase activity was measured as described in the methods. The luciferase activity of the treated samples is expressed relative to the activity of untreated control (no avicin, no IL-6), which is taken as 100%. (B) Effect of avicin D on IL-6 production in U266 cells. Cells were treated with 0–1 µM of avicin D for 48 hrs. Cell supernatants were assayed for IL-6 levels using an ELISA kit. (C) Effect of avicin D on the expression of Stat3 regulated proteins. U266 cells were treated with 1 µM of avicin D for 0–24 hrs. Whole cell lysates were analyzed for the expression of different proteins by western blot analysis as described in the methods.