Figure 4. Contribution of E-cadherin and β-catenin to 2-ME₂-mediated effects on Bic-1 cell motility.

A. Representative images of the scratch test show untreated Bic-1 cells grown in serum-free conditions (control) at 0 h or 24 h after scratch and 2-ME₂ treated Bic-1 cells at similar time points. In the control test the distance between the borderlines becomes infiltrated with motile Bic-1 cells 24 h after scratch, while it is still preserved in 2-ME₂ treated samples (middle panel). Immunofluorescence images (right panel) of GFP-tagged Bic-1 cells in 2-ME₂-treated and untreated cultures.

B. Reduced expression of E-cadherin (left panel) and β-catenin (right panel) by gene-directed shRNAs. E-cadherin level was determined by Western blotting in mismatched shRNA transfected and E-cadherin shRNA-transfected Bic-1 cells. β-catenin protein expression in Bic-1 cells was analyzed by immunoflorescence technique using a monoclonal antibody against β-catenin (red). Nuclei are counterstained with DAPI (blue). a. mismatched-shRNA transfected cells, b. β-catenin-shRNA transfected cells and c. β-catenin-shRNA transfected cells and 2-ME₂ (5μM) treated cells.

C. Contribution of E-cadherin (left panel) and β-catenin (right panel) to 2-ME₂-mediated effects on Bic-1 cell migration. E-cadherin or β-catenin-transfected Bic-1 cells were placed in the upper chamber of Boyden chamber in the presence or absence of 2-ME₂ and cell migration towards serum was allowed to proceed for 24 h. Treatment series are: control (1), control+2-ME₂ (2), scrambled control (3), scrambled control+2-ME₂ (4), E-cadherin/β-catenin-shRNA (5) and E-cadherin/β-catenin-shRNA+2-ME₂ (6). The results reflect the mean (± SD) number of migrated cells to the undersurface of the matrigel membrane from 3 independent experiments. *p<0.05 vs controls; **p<0.01 vs shRNA; ***p<0.001 vs control/scrambled control.