Cell Culture and Transfection Protocols

MCF7, MDA-MB-231, MDA-MB-LM2 (LM2) and HEK293T cells (ATCC) were cultured in DMEM (4.5 g/liter glucose) with 10% FBS and 20 mM Hepes, pH 7.2. MCF10a cells were cultured in DMEM:F12 50/50 with 5% FBS, 0.5mg/mL hydrocortisone, 100ng/mL cholera toxin and 20ng/mL EGF. MCF-7, MCF-10A, MDA-MB-231 and LM2 cells were transfected with 10μg DNA using the Amaxa^TM nucleofector (Lonza) as recommended. HEK293T cells were transfected using JetPrime^TM (Polyplus Transfection, Illkirch, France) as recommended. Microinjection was performed as described [40 (link)]. Briefly, 5-20 μg/ml cDNAs in HKCl (10 mM Hepes, 140 mM KCl, pH 7.4) were pressure injected into cell nuclei using a micromanipulator (MMO-202ND; Narishige). Cells were incubated at 37°C for 90min to allow for expression of cDNAs.