MCF10CA1h cells were grown in a 1∶1 mixture of DMEM and Ham's F12 medium (Gibco) supplemented with 5% horse serum. Cells were collected using trypsin and transfected using an Amaxa Nucleofector device using solution V and program T-27 according to manufacturer's procedures. 2.0×106 cells were transfected with 400 nmoles siRNA duplex (Darmacon) or a GFP-Max nucleofection control. Post nucleofection 1.0×106 cells were plated to each well of a 6 well tissue culture plate. Cells were grown for 2 days in growth media and then serum deprived in 1% FBS, DMEM, nonessential amino acids, 10 µM β-mercaptoethanol overnight. Following serum shock cells were incubated with or without 5 ng/ml TGF-β1 (R&D Systems) for 1 hour. Cells were lysed using TriReagent reagent (Sigma) and RNA and protein were purified according to manufacturer's procedures.
