Fig. 4. Cyclin D1 half-life is not regulated by E-cadherin. MCF10A cells were transfected with control or E-cadherin siRNA during serum and growth factor starvation. (A) The starved cells were trypsinized, reseeded in suspension on agarose-coated dishes and stimulated with 10% FBS and growth factor cocktail for 6 hours to induce cyclin D1. Cycloheximide (CHX) was then added, and the cells were collected every 15 minutes for 1 hour. Collected cells were analyzed by western blotting using anti-cyclin D1 and anti-GAPDH (loading control). The cyclin D1 blot was exposed for 10 and 30 seconds (s) so that all bands could be readily detected and easily compared between samples (e.g. compare 10 s `control siRNA' with 30 s `E-cadherin siRNA'). (B) MCF10A cells were transfected with control or E-cadherin siRNA and then infected with β-galactosidase (LacZ) (20 MOI) or cyclin D1 adenoviruses (2.5-20 MOI) during serum and growth factor starvation. The cells were trypsinized, reseeded in monolayer [M; β-galactosidase control only] or suspension (Susp) on dishes coated with collagen or agarose, respectively. The cells were stimulated with 10% FBS and growth factor cocktail for 9 hours. Cell lysates were analyzed by western blotting for cyclin D1, E-cadherin, and actin (loading control).