Supplemental Material

This article contains the following supporting material:

• Figure 1 - Reduction in E-cadherin functions using siRNA. (A) COS-7 cells were co-transfected with the E-cadherin expression construct and siRNA constructs (Ecad-siRNA1, Ecad-siRNA2, and Ecad-siRNA3, targeting different sequences of E-cadherin), or the vector pSuper to examine the efficacy of silencing of the siRNA constructs. Untransfected COS-7 cells (COS-7) and COS-7 cells transfected with the E-cadherin expression construct (control) were included in the Western blot analysis probed with an anti-E-cadherin antibody. The same membrane was re-probed with anti-b-actin antibody for loading assessment. After development, the films were overlapped and photographed. All three siRNA constructs reduced E-cadherin expression. (B) The E-cad-siRNA1 construct was co-transfected into V1-expressing cells with the plasmid pcDAN3.1/hygro. Stably transfected cell lines were selected using hygromycin at 0.3 mg/ml. Inhibition of E-cadherin by siRNA was examined by RT-PCR. Results from two typical cell lines (Ecad-siRNA1a and Ecad-siRNA1b) are shown. V1-transfected (V1) and vector-transfected (vector) NIH3T3 fibroblasts served as positive and negative controls. Reduced E-cadherin expression was observed in the siRNA-transfected cells. (C) Cell lysate was also analyzed on Western blot for E-cadherin expression. Equal loading was confirmed by probing the membrane with an anti-actin antibody.

• Figure 2 - Reversion of cell behavior through siRNA targeting E-cadherin expression. (A) siRNA-mediated silencing of E-cadherin was examined by immunostaining. Little E-cadherin signal was detected in siRNA-transfected cells compared to the vector control (pSuper). E-cadherin silencing induced translocation of b-catenin and p120 to cell surface. Images were captured with a confocal microscope (scale bar, 25 μm). (B) Cell morphology was examined under a light microscope. Transfection of Ecad-siRNA1a induced cell dissociation compared to pSuper-transfected cells (scale bar, 150 μm).