Figure 6.

Vsnl1 suppresses canonical WNT signaling activity in mIMCD3 cells. (A) Luciferase reporter assay in mIMCD3 cells. The cells were transiently cotransfected with TOPFLASH and Vsnl1 cDNA or empty vector and cultured in Wnt3A conditioned medium or control medium for 2 hours. Luciferase activity was reduced in Vsnl1-transfected cells (about 50%; P < 0.005) grown in both control and Wnt3A-conditioned medium (about 30%; P < 0005). (B) Western blot analysis. Western blotting of mIMCD3 cells lysates was done for phosphoSer9-GSK3β, total GSK3β, activated β-catenin, and total β-catenin. β-Actin served as a loading control. Vsnl1 increased the total GSK3β levels and decreased the levels of inactive phosphoSer9-GSK3β when compared with controls. (C) Quantitative analysis. (relative activity/β-actin) of the Western blots results revealed a decrease of the total level of β-catenin in both control (about 30%, P < 0005) and Wnt3A medium (24%, P < 0005). In Vsnl1 transfected cells, total GSKβ level was increased in non-Wnt3A-treated cells by 2.9-fold and by 1.4-fold in Wnt3A-stimulated cells. Respectively, the inactivated ser9-phospho GSK3β shows a 5-fold decrease in control medium and a 2.5=fold decrease in Wnt3A-stimulated cells, when compared with mock transfections (B and C). The fold changes represent the averages ± SE in three independent experiments. ***P < 0.005.