Fig. 3. E-cadherin-mediated cell-cell adhesion and cell-substratum signaling coordinately regulate cyclin D1 expression in MCF10A cells. MCF10A cells were transfected with control or E-cadherin (E-cad) siRNA, rendered quiescent by serum and growth factor starvation, trypsinized, and reseeded in a monolayer (Mono) or in suspension (Susp) on collagen- or agarose-coated dishes, respectively. The cells were stimulated with 10% FBS and growth factor cocktail. (A) Samples were collected at the indicated time points and analyzed by western blotting for cyclin D1, Cdk6, E-cadherin, β-catenin, phosphoY397-FAK and FAK. (B) The experiment in A was repeated with cells seeded in monolayer (on collagen-coated dishes containing glass coverslips) (Liu et al., 2006) or in suspension (on agarose-coated dishes) at 2x10⁴ cells/cm². Collected cells were analyzed by QPCR for cyclin D1 mRNA. (C) Cells seeded on collagen-coated dishes and stimulated with 10% FBS and growth factor cocktail were collected and analyzed by western blotting for E-cadherin, p21Cip1, p27Kip1, dually phosphorylated ERK, total ERK and GAPDH (loading control).