L glutamine

BC3, BCBL1 (human B-cell lines derived from PEL carrying latent KSHV), Raji (human B cell line derived from BL carrying latent EBV) and B95-8 (marmoset B-cell line EBV-infected)^{31 (link)} were cultured in RPMI 1640 (Sigma Aldrich), 10% Fetal Bovine Serum (FBS) (Sigma Aldrich), L-glutamine (Aurogene) and streptomycin (100 μg/ml) and penicillin (100 U/ml) (Aurogene) in 5% CO₂ at 37 °C.

Human B cell lines derived from KSHV-positive PEL cell lines, BC3, and BCBL-1 (kindly supplied by Prof. P. Monini, National AIDS center, Istituto Superiore di Sanità, Rome, Italy) were grown in RPMI 1640 medium (Sigma-Aldrich, Burlington, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Burlington, MA, USA), L-glutamine (2 mM) (Aurogene, Rome, Italy), and streptomycin/penicillin (100 μg/mL) (Aurogene, Rome, Italy) (complete medium) at 37 °C in a 5% CO₂ humified setting. Cells were seeded into 6-well plates at a density of 4 × 10⁵ per well in a final volume of 2 mL in complete medium and were treated for 24 h (h) singly or in combinations with the STAT3 inhibitor tyrphostin AG490 (50–100–200 µM) (Calbiochem, San Diego, CA, USA; 658411) and the NFE2L2 inhibitor brusatol (10–20–40 nM) (Sigma Aldrich, St. Louis, MO, USA; 1868). In some experiments, to evaluate the role of HSP90A and HSPB1 in the mechanism/s to which NFE2L2 could sustain STAT3 activity and vice versa, PEL cells were treated for 24 h with inhibitors of HSPB1 and HSP90A, respectively, J2 (10 µM) (MedChemExpress, Monmouth Junction, NJ, USA; HY-124653), 17-AAG (0.1 µM) (Selleckem, Planegg, Germany; S1141). All the drugs were dissolved in DMSO, and the control cells were supplemented with DMSO in the same amount used for the other samples.

Human colon carcinoma Caco-2 cells (ATCC, HTB-37 clone) were routinely cultured in 25 cm² culture flasks (BD Biosciences, New Jersey, USA) in Dulbecco’s modified Eagle’s medium (DMEM) at pH 7.4. Supplementation was with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% (v/v) HEPES 1M, 1% (v/v) non-essential amino acids (Gibco), 1% L-glutamine 200 mM, penicillin (100 U/mL), and streptomycin (100 mg/mL) (Aurogene, Italy). Cells stayed in an incubator at 37 °C in a 5% CO₂ and 95% air-humidified atmosphere. Refreshing of the medium was every 2–3 days and cells were split once a week. Cells were observed periodically for changes in growth by using an inverted-phase contrast microscope (Olympus, CK2 model). For cell treatments, freeze-dried digested pasta samples were resuspended in DMEM (10 mg/mL, stock solution) and sterilized through 0.22 mm filter membrane (Millipore Corporation, Bedford, MA, USA).