Mebm mammary epithelial cell growth medium

All cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (Life Technologies). The immortalized nontumorigenic HMECs were cultured in 1/2 MEBM Mammary Epithelial Cell Growth Medium from Lonza (Allendale, NJ, USA) and 1/2 DMEM/F12 supplemented with insulin (5 μg/ml), EGF (10 ng/ml), and hydrocortisone (0.5 μg/ml) as described before.^{42 (link)} For cell viability measurement, cells were seeded in 96-well plates at a density of 3000 cells per well in 100 μl medium containing either 25 or 1 mM glucose. When needed, PLD1i or PLD2i was added to the medium at the final concentration of 5 μM. In some experiments, cells were also treated with different concentrations of 2-DG (0.25, 0.5, 1, 2.5, and 5 mM), 20 μM oleic acid (Sigma-Aldrich), 200 μM etomoxir (Sigma-Aldrich), 1 μM methyl-pyruvate (Santa Cruz Biotechnology, Dallas, TX, USA), or 0.1 mM NAC (Sigma-Aldrich). The numbers of viable cells were measured at indicated time points by CyQuant Cell Proliferation Assay Kit (Life Technologies).

MCF10A cells (#CRL-10317,) were purchased from ATCC (Manassas, VA, USA) and cultured in MEBM mammary epithelial cell growth medium (#CC-3151) supplemented with bovine pituitary extract, human epidermal growth factor, hydrocortisone, insulin, and gentamicin SingleQuots (#CC-4136) (Lonza, Walkersville, MD, USA) and maintained at 37 °C in an atmosphere of 5% CO₂ in room air. MCF10A cells were plated at a density of 400,000 cells per 60 mm cell culture dish and grown overnight. The cells were then maintained for three weeks in complete media containing the NO donor DETANO at concentrations of 0, 100, 300, and 500 μM. The media was changed each Monday and Friday and fresh DETANO was added. During this treatment period, the cells were passaged as necessary to avoid confluence. At the end of the three-week treatment, DETANO was removed, and the cells were examined for phenotypic changes.