JCS017012 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Distinct E-cadherin siRNAs similarly regulate cyclin D1 levels. MCF10A cells were transfected with control or two distinct E-cadherin siRNAs, rendered quiescent by serum and growth-factor starvation, trypsinized and reseeded in monolayer (ML) or suspension (Sus) on collagen- or agarose-coated dishes, respectively. The cells were stimulated with 10% FBS and growth factor cocktail. Samples were collected at the indicated times and analyzed by western blotting for cyclin D1, actin (loading control), E-cadherin, β-catenin, phosphoY397-FAK and FAK.

• Supplemental Figure S2 -

  Fig. S2. Effects of dominant-negative E-cadherin on MCF10A morphology and cyclin D1 mRNA. MCF10A cells were infected with adenoviruses encoding β-galactosidase (LacZ) or a dominant-negative E-cadherin lacking the cytoplasmic domain (EcadΔcyt; kind gift of Meenhard Herlyn, Wistar Institute) at 300 MOI and serum starved (see Materials and Methods). The cells were trypsinized, replated in monolayer or suspension culture (on collagen- or agarose-coated dishes, respectively), and stimulated with 10% FBS and growth factors for 9 hours. (A) Cells plated on collagen were stained with anti-E-cadherin, anti-β-catenin and DAPI (nuclei). Bar, 40 µm. Note the loss of E-cadherin and β-catenin at cell-cell junctions in response to the dominant-negative E-cadherin. (B) Collected cells were analyzed by QPCR for the level of cyclin D1 mRNA (dn-Ecad, dominant-negative E-cadherin).