Non essential amino acids (neaa)

Drosophila melanogaster cells (JW18) with and without Wolbachia (strain wMel) and Aedes albopictus cells (C710) with and without Wolbachia (strain wStri) were grown at 24 °C in Shields and Sang M3 insect media (Sigma-Aldrich, Burlington, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Waltham, MA, USA), 1% each of l-glutamine (Corning, Corning, NY, USA), non-essential amino acids (Corning, Corning, NY, USA) and penicillin-streptomycin-antimycotic (Corning, Corning, NY, USA). Baby hamster kidney fibroblast (BHK-21) cells were grown at 37 °C under 5% CO₂ in 1× Minimal Essential Medium (Corning, Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Corning, Corning, NY, USA), 1% each of l-glutamine (Corning, Corning, NY, USA), non-essential amino acids (Corning, Corning, NY, USA) and penicillin-streptomycin-antimycotic (Corning, Corning, NY, USA).

The Abrams cell line was maintained in DMEM supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, penicillin, streptomycin and L-glutamine (Corning). OSA8, OSA16 and D17 cell lines were maintained in RPMI supplemented with 10% FBS, 0.1 mM non-essential amino acids, sodium pyruvate, penicillin, streptomycin, L-glutamine (Corning). Cells were maintained in monolayer culture at 37°C in humidified air with 5% CO2 in these growth media.

Caki-1 and Caki-2 cells (American Type Culture Collection, Manassas, VA) were cultured in McCoy’s 5A (Corning Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (Omega Scientific,Inc, Tarzana, CA), 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Corning Mediatech, Inc., Manassas, VA), 1% nonessential amino acids (Corning Mediatech, Inc., Manassas, VA) and 1% L-glutamine (Corning Mediatech, Inc., Manassas, VA) at 37°C in a humidified atmosphere in a 5% CO2 incubator. A704 and A498 cells (American Type Culture Collection, Manassas, VA) were cultured in Eagles Minimum Essential Media supplemented with 10% fetal bovine serum (Omega Scientific,Inc, Tarzana, CA), 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; Corning Mediatech, Inc., Manassas, VA), 1% nonessential aminoacids (Corning Mediatech, Inc., Manassas, VA) and 1% L-glutamine (Corning Mediatech, Inc., Manassas, VA). All cells were maintained at 37°C in a humidified atmosphere in a 5% CO₂ incubator.

MCF-7 and T47D cells were cultured in growth medium containing Dulbecco's minimum essential medium (DMEM) (Cellgro by Mediatech Inc, Manassas, VA) plus 10% fetal bovine serum (FBS) (Hyclone by Thermo Scientific, South Logan, UT), and 1% each of nonessential amino acids (NEAA) (Cellgro), Glutamax and Penicillin-streptomycin-neomycin (PSN) antibiotics mixture (Gibco by Invitrogen Corp. Carlsbad, CA) and maintained at 37°C and 5% CO₂. For each experiment, MCF-7 cells are seeded in growth medium for 24 hr. The medium was then replaced with steroid-free medium containing phenol red-free DMEM plus 10% charcoal/dextran-stripped (cs) FBS, and 1% each of NEAA, Glutamax and PSN, and the cells were incubated at 37°C for 48–72 hr before treatment. The cells were pre-treated with 1 μM ICI 182,780 (ICI) or 1 μM in solution AMPK inhibitor compound C/Dorsomorphin (DOS) (Calbiochem, EMD Millipore Corp. Billerica, MA). The cells were treated simultaneously with the following, unless otherwise indicated: 10 ng/ml human tumor necrosis factor alpha (TNFα; Invitrogen), and 10 nM E2, 10 μM resveratrol (RES), 25 μM Rolipram (ROL), or 10 μM Forskolin (FSK) (Sigma–Aldrich Inc., St. Louis, MO).