Fig. 2. E-cadherin-mediated adhesion in suspended MCF10A cells. (A) Quiescent MCF10A cells were trypsinized, reseeded on dishes coated with agarose, stimulated with 10% FBS and growth factor cocktail for 9 hours, and analyzed by phase-contrast or epifluorescence microscopy for E-cadherin (E-cad) and β-catenin (β-cat). (B) MCF10A cells were transfected with control or E-cadherin siRNA, rendered quiescent by serum and growth factor starvation, trypsinized, reseeded on collagen-coated dishes, stimulated with 10% FBS and growth factor cocktail for 9 hours, and analyzed by epifluorescence microscopy for E-cadherin, β-catenin and DAPI-stained nuclei. (C) Cells prepared as in B were seeded on agarose-coated dishes with 10% FBS and growth factor cocktail for 9 hours, collected on poly-L-lysine-coated coverslips, and analyzed by phase-contrast microscopy. (D) Quiescent MCF10A cells were trypsinized and pre-incubated in suspension in the absence and presence of 2 mM EGTA (30 minutes at 37°C) in DMEM-F12 with 1 mg/ml heat-inactivated fatty acid-free BSA. The pre-treated cells were directly replated on agarose-coated dishes, and stimulated with 10% FBS and growth factor cocktail for 9 hours. The cells were then collected on poly-L-lysine-coated coverslips and analyzed by phase-contrast microscopy. Bars, 210 µm.