Non essential amino acids (neaa)

All cell lines were obtained from ATCC, except Jurkat cells, which were a gift from Professor Holley, University of Sheffield. MCF-7, MDA-MB-231, MDA-MB-468, and T-47D cell lines were grown in DMEM containing 4.5 g/L glucose with L-glutamine, 10% FCS (Seralab) and 1X nonessential amino acids (Bio Whittaker). Jurkat cells were grown in RPMI 1640 (Lonza) containing L-glutamine, 10% FCS and 1X nonessential amino acids. MCF-10A cells were grown in DMEM containing 4.5 g/L glucose with L-glutamine with the addition of 1X nonessential amino acids, 5% horse serum (Invitrogen), 10 µg/mL insulin (Sigma-Aldrich), 0.1 µg/mL cholera toxin (Calbiochem), 10 µg/mL epidermal growth factor (EGF; Sigma-Aldrich), and 50 µM hydrocortisone (Sigma-Aldrich). All cell lines were used within 20 passages and regularly checked for Mycoplasma.

Huh7, HEK 293T cells stably expressing TLR3 (generous gifts from Dr. Kate Fitzgerald), HEK 293T (Dharmacon, Inc.), and Vero (obtained from American Type Culture Collection (ATCC)) cells were maintained in Dulbecco’s modified minimal essential medium and HepG2 (ATCC) cells were maintained in Eagle’s Minimum Essential Medium, both supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich), 1% non-essential amino acids (Lonza) and 1% L-Glutamine solution (Sigma-Aldrich). U937 cells stably expressing DC-SIGN (a generous gift from Dr. Anuja Mathew) were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich), 1% non-essential amino acids (Lonza), and 1% L-Glutamine solution (Sigma-Aldrich). All cells were incubated in a humidified chamber at 37°C and 5% CO2. DENV2 strains DENV2 16681 and NGC were originally obtained from ATCC or the Walter Reed Army Institute of Research and were passaged in C6/36 cells (ATCC). Virus titers were determined by immunostained plaque assay on Vero cells (Liu et al., 2012 (link); Medin et al., 2015 (link)).

Lymph node cells were obtained by mechanical separation of lymph nodes and frozen at 5 × 10⁶ cells/ml in a solution of 90% FBS and 10% DMSO with 2.5 μg/ml Amphotericin B (Lonza). Cells were stored in liquid nitrogen until use, then thawed and resuspended at 10⁶ cells/ml in complete RPMI 1640 medium supplemented with L-Glutamine, sodium pyruvate, HEPES, non-essential amino acids (Lonza), 10% heat-inactivated FBS (Hyclone), and IL-2 at 5 ng/ml (PeproTech). Phytohemagglutinin at 10 µg/ml (Sigma-Aldrich, St Louis, MO) was added to activate cells. PBMCs were isolated by density gradient centrifugation using Histopaque 1077 (Sigma-Aldrich) and cultured at 10⁶ cells/ml in complete RPMI 1640 medium supplemented with L-Glutamine, sodium pyruvate, HEPES, non-essential amino acids (Lonza), 10% heat-inactivated FBS (GE Healthcare Bio-Sciences, Pittsburgh, PA), and IL-2 at 5 ng/ml (PeproTech, Rocky Hill, NJ). Phytohemagglutinin at 10 µg/ml (Sigma-Aldrich) was added to activate cells. For both primary cell types, donor cells for coculture infection were cultured for one day then infected by cell-free virus, while target cells were cultured for three days and infected with either cell-free HIV or infected donor cells.

Madin-Darby canine kidney (MDCK) cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Greiner), 100 U/ml penicillin (PEN) (Lonza), 100 U/ml streptomycin (STR) (Lonza), 2 mM l-glutamine (l-Glu) (Lonza), 1.5 mg/ml sodium bicarbonate (NaHCO₃) (Lonza), 10 mM HEPES (Lonza) and 1× nonessential amino acids (NEAA) (Lonza). 293T cells were cultured in Dulbecco modified Eagle’s medium (DMEM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 U/ml STR, 2 mM l-Glu, 1 mM NaHCO₃, and 1× NEAA. Vero cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 mg/ml STR, and 2 mM l-Glu.

HCT-116 and Caco-2 cell lines, both derived from primary tumors, were obtained from the Interlab Cell Line Collection (ICLC), an International Certified Repository Authority within the IRCCS Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro (Genova, Italy). HCT-116 cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine serum (FBS) (Gibco) and 2 mM L-glutamine (Lonza, Basel, Switzerland); Caco-2 cells were cultured in DMEM (Sigma-Aldrich, St. Louis, MO), supplemented with 20% FBS (Gibco), 2 mM L-glutamine (Lonza), and 1% non-essential amino acids (NEAA) (Lonza); both media were supplemented with 1% penicillin/streptomycin (10,000 U/mL) (Gibco). Cells were cultured at 37°C and 5% CO₂.

Human HepG2 cells (ECACC 85011430) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and cultured in Eagle’s Minimum Essential Medium (EMEM) without L-glutamine (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM glutamine (EuroClone, Pero, Italy), 0.1 mM Non-Essential Amino Acids NEAA (Lonza), 100 U/ml penicillin and 100 μg/ml streptomycin (EuroClone). The cells were incubated at 37 °C in 5% CO₂. For the treatments, HepG2 cells were transferred into 12-well plates (1.0 × 10⁵ cells per well) and incubated for overnight. The cells were subsequently treated with 500 μM OA (O2750, Sigma-Aldrich), 50 ng/ml LPS (L5543, Sigma-Aldrich) or with these both for 5 days in the culture medium. OA was dissolved in DMSO, from which the working stock was prepared by diluting it in cell culture medium. Solvent controls were included in the treatments. After 48-h incubation, the culture medium was replaced with newly-prepared medium and the incubation proceeded for 5 days.