Mammary epithelial cell growth medium

Cells were cultured under constant humidity at 37 °C and 5% CO₂. MCF10A cells were from ATCC (Manassas, VA, USA) and cultured with DMEM/Ham's F12 containing Horse serum 5%, EGF 20 ng ml⁻¹, Insulin 5 μg ml⁻¹, Hydrocortisone 0.5 μg ml⁻¹, Choleratoxin 0.1 μg ml⁻¹ and penicillin/streptomycin (P/S). MCF7 cells were from DSMZ (Braunschweig, Germany) and cultured with RPMI 1640 containing FCS 10%, Insulin 5 μg ml⁻¹, Pyruvate 1 mM, NEAA 1% and P/S. The human urinary carcinoma cell line T24 was kindly provided by Dr B Mayer (Surgical Clinic, LMU, Munich, Germany). T24 were cultured with McCoyś medium containing FCS 10%, glutamine (1.5 mM) and for lentiviral-transduced T24 cells, puromycin (1 μg ml⁻¹) was used. Immortalised human mammary epithelial (HMLE) cells stably transfected with Twist-ER were described previously (Scheel et al, 2011 (link)). In brief, HMLE cells were cultivated in mammary epithelial cell growth medium (PromoCell GmbH, Heidelberg, Germany) supplemented with P/S (PAA Laboratories, Pasching, Austria) and 10 μg ml⁻¹ blasticidin (Gibco, Germering, Germany). To induce EMT, HMLE Twist-ER cells were treated with 20 nM 4-Hydroxytamoxifen (4-OH-TX; Sigma-Aldrich, Taufkirchen, Germany) for 10 days, whereby cells were split and supplied with fresh medium and stimulation reagents every 3 days.