Supplemental Material

This article contains the following supporting material:

• Figure 1S - β-catenin binding domain of E-cadherin is necessary for inhibition of MDA-MB-231 cell proliferation by E-cadherin ligation. Effects of Fc-hE coated microsphere binding to cell surfaces on cell proliferation of isolated MDA-MB-231 cells expressing wild-type (A) and mutant forms of E-cadherin ((B) E-cadherin Δβ-catenin, (C) E-cadherin/α-catenin fusion, (D) E-cadherin Δ p120^ctn) as determined by BrdU incorporation when compared with ligation control. Cells were grown in presence of 1 μg/ml of doxycycline as previously described by Wong and Gumbiner, 2003. Data are expressed as mean ± SEM (*p<0.05).
• Figure 2S A-E - Specific rescue of three different anti-β-catenin siRNAs by specifically mutated forms of β-catenin. (A) Cartoon representing full length β-catenin and location of the three siRNA targets (bold and underlined characters). Bold, underlined and italic characters represent neutral mutations in β-catenin DNA sequence obtained by directed mutagenesis. (B, D, F) MCF-7 cells and (C, E, G) SW480/E-cadherin FL stable clone. (B-C) Western blot of β-catenin in lysates from cells co-transfected with siRNAs against GFP or β-catenin (β-cat#1, β-cat #2 and β-cat #3) and empty pcDNA3 or expression vectors coding β-catenin mutated at the siRNA target sites (β-cat FL #1, β-cat FL #2 and β-cat FL #3). (D-E) Indirect immunofluorescence staining of β-catenin in cells co-transfected with siRNAs against GFP or catenin and expression vector coding β-catenin mutated for the different siRNA targets.
• Figure 2S F-G - (F-G) E-cadherin ligation inhibits the proliferation of cells only when β-catenin expression specifically rescues siRNA depletion of β-catenin. The specific β-catenin siRNA used is indicated at the top of each bar graph, and the specific β-catenin mutant used to rescue expression is indicated at the left side. Data are expressed as mean ± SEM (*p<0.05).
• Figure 3S - Western Blots showing the specificity of the phospho-specific antibodies. A431 cells were serum starved overnight and stimulated without or with 10 ng/ml EGF for the indicated time periods. Then cells were lysed with RIPA buffer and the whole cell lysates were analyzed by western blotting for (A) phosphorylated-EGFR (Tyr1173) and phosophorylated-EGFR (Tyr845); (B) phosphorylated-ERK and (C) phosphorylated-STAT5.