Mammary epithelial growth supplement

Human malignant luminal ER⁺/PR⁺/HER2⁻ (T47D), basal-like ER⁻/PR⁻/HER2⁻ (MDA-MB-231) and non-malignant (MCF10A) breast cells were chosen to represent three different breast cell subtypes. Cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). T47D cells were maintained in growth medium containing, RPMI 1640 (SIGMA, St. Louis, MA) supplemented with fetal bovine serum (10 %), insulin (0.2 units/mL), sodium pyruvate (1.0 mM) and penicillin/streptomycin (1 %). MDA-MB-231 cells were maintained in L15 medium containing penicillin/streptomycin (1 %) and horse serum (10 %). MCF10A cells were maintained in 171 Medium supplemented with Mammary Epithelial Growth Supplement (Invitrogen, Carlsbad, CA). All culture mediums contained ~5 μM Zn as assessed by atomic absorption spectroscopy. Cells were routinely cultured in plastic 75 cm² flasks and sub-cultured every 4–5 days. Cells were maintained in a humidified chamber in 5 % CO₂ at 37 °C.

Liebovitz’s L15 Medium and 0.5% Trypsin Phosphate Versene Glucose (TPVG) was purchased from HiMedia (USA). M171 Medium and Mammary Epithelial Growth Supplement (MEGS) were obtained from Life Technologies (USA). Fetal Bovine Serum (FBS), Penicillin, and Streptomycin (P&S) were purchased from Gibco (Grand island, NY, USA). All reagents were procured from Sigma-Aldrich (St. Louis, MO, USA) except for if otherwise mentioned. Primary antibodies for Immunofluorescence and Western Blotting were purchased from Cell Signaling Technologies (CST), USA and Santa Cruz Biotechnology (Santa Cruz, CA, USA). AlexaFluor secondary antibodies for Immunofluorescence and Flow Cytometry were obtained from Invitrogen (Life Technologies, USA). HRP-Linked anti-rabbit or anti-mouse secondary antibodies for Western Blotting were obtained from BioRad. All ELISA kits were purchased from R&D Systems, USA.

BT474 cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). The BT474 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Wisent) with 10% fetal bovine serum (FBS, Wisent) and antibiotics (Penicillin/Streptomycin, 100 units/ml; Life Technologies). The primary BM-132 cells were derived from a HER2+ breast cancer patient pre-treated with chemotherapy and developed resistance to Trastuzumab. The BM-132 cells were grown in 66% DMEM high glucose (Wisent), 25% Ham’s F12 nutrient mix (Invitrogen), 7.5% FBS (Wisent), 0.4 ug/ml Hydrocortisone (Stem cell technologies), 5 ug/ml Insulin (Sigma), 8.4 ng/ml Cholera Toxin (Sigma), 10 ng/ml EGF (Invitrogen), 10 µM Y-27632 RHO/ROCK pathway inhibitor (Stem cell technologies), 100 unit/ml Penicillin and 100 unit/ml Streptomycin (Gibco), 1.48 mM L-glutamine (Wisent). The mouse NEU tumor cells were maintained in the same media supplemented with Mammary Epithelial Growth Supplement (Life Technologies) consisting of 0.4% v/v bovine pituitary extract (BPE), 1 µg/ml recombinant human Insulin-like growth factor 1, 0.5 µg/ml Hydrocortisone and 3 ng/ml human epidermal growth factor. Cells were tested for mycoplasma contamination. SAL003 was purchased from Sigma-Aldrich (# S4451) whereas Trastuzumab was manufactured by Roche and provided by the Pharmacy of Jewish General Hospital (JGH).

All mammary tumor cell lines were maintained in DMEM/F12 containing 10% FBS, 1% mammary epithelial growth supplement (Life Technologies), 1% penicillin/streptomycin, and 1% l-glutamine. Mammary tumor cell lines were isolated as described from individual tumors at endpoint [23 (link)]. All experiments were performed on three independent isolates and representative data are shown. All mammary tumor cell lines were used within 30 passages of initial isolation. All other cell lines used were maintained in DMEM containing 10% FBS, 1% penicillin/streptomycin, and 1% l-glutamine. Cells were cultured at 37°C in a humidified incubator set at 5% CO₂. Cells were routinely passaged at a one in ten dilution every 2–3 days.
For plasmid and siRNA (Supplementary Table 3) transfections, cells were seeded such that they would reach approximately 75% confluency the day of the transfection. Lipofectamine 3000 was used for all transfections according to the manufacturer’s protocol using 8 μg of plasmid DNA or 200 nM of each siRNAs. Transfections were performed for 48–72 h prior to cell harvest.