Glutamine

In this experiment, a male patient-derived human dermal microvascular endothelium (HMEC-1) cell line was utilized. The HMEC-1 cells (#CRL-3243) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in MCDB131 (#10372019, ThermoFisher Scientific, Waltham, MA, USA) complete growth medium supplemented with 10% fetal bovine serum (FBS; #30-2020, ATCC, Manassas, VA, USA), 10 ng/mL epidermal growth factor (EGF; #PHG0314, ThermoFisher Scientific, Waltham, MA, USA), 1 µg/mL hydrocortisone (#H0396, Sigma, St. Louis, MO, USA), 10 mM glutamine (#302214, ATCC, Manassas, VA, USA), and 100 units/mL of penicillin and 100 µg/mL of streptomycin (#15140122, ThermoFisher Scientific, Waltham, MA, USA) at 37 °C under a humid atmosphere of 5% CO₂/95% air, as per the manufacturer’s instructions. The complete growth medium was refreshed twice per week.
For irradiation (IR) experiments, HMEC-1 cells (at passage 7) were seeded in T25 flasks at a density of approximately 3 × 10⁶ cells per flask and cultured for three to four days prior to IR. The day before IR, when the cultures were in the early plateau phase, the complete growth medium was substituted with fresh cell starvation medium, which contained MCDB131, 0.1% FBS, 1 µg/mL hydrocortisone, 10 mM glutamine, and 100 units/mL of penicillin and 100 µg/mL of streptomycin.

The previously described F9 and dyskerin-mutated F9 cells (F9-A353V) [19 (link), 21 (link)], were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum, 2 mM glutamine (Gibco) and 1.5 gr/ml of sodium bicarbonate. HEK293T cells (ATCC) were cultured in DMEM media supplemented with 10% foetal bovine serum and 2 mM glutamine. DC-C and DC-3 cells (Coriell Cell Repository) were cultured in Roswell Park Memorial Institute (RPMI) media supplemented with 20% foetal bovine serum and 2 mM glutamine. XDC-1787-C (Corriel Cell Repository) and F26IIB cells (generated in our laboratory), were cultured in Minimum Essential Medium Eagle (MEM) media supplemented with 15% foetal bovine serum and 2 mM glutamine. Cells were transfected with 10 μg of DNA/10⁶ cells using lipofectamine plus (Invitrogene, Carlsbad, USA) or the K2 transfection kit (Biontex Lab, Planegg/Martinsried, GE) according to the manufacturers’ instructions. 15 μg of peptides per 35 mm dish were transfected using the ProteoJuice Protein Transfection Reagent (Merck Millipore, Billerica, MA, USA) or the K2 transfection kit.

The materials were sterilized by UV light for 0.5 h from both sides. The samples were inserted into polystyrene 24-well cell culture plates (TPP, Trasadingen, Switzerland) and were seeded with endothelial cells originating from bovine pulmonary artery (line CPAE ATCC CCL-209, Rockville, MA, USA) in a minimum essential Eagle medium (E-MEM) supplemented with 2 mM l-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential aminoacids and 1.5 g/L sodium bicarbonate (all chemicals from Sigma). In order to avoid potential adsorption of serum-derived proteins and masking the oligopeptidic ligands, fetal bovine serum (Sebak GmbH, Aidenbach, Germany) was added to the medium 5 h after cell seeding to a final concentration of 20 % in each well [23 (link)]. Each well contained 30,000 cells and 1.5 ml of the medium. The cells were cultured in a humidified atmosphere containing 5 % CO₂ in the air.

T. gondii lines used include GFP-expressing RH (type I), ME49/PTG (type II) and RFP-expressing PRU or ME49 (type II) (Kim et al., 2001 (link); Hitziger et al., 2005 (link)). N. caninum lines used include NC-1 and NC-Liverpool (ATCC 50977 and ATCC 50845, American Type Culture Collection, Manassas, Virginia, US). Tachyzoites were maintained by serial 2-day passaging in human foreskin fibroblast (HFF-1, ATCC SCRC-1041) monolayers cultured in DMEM (Thermofisher scientific, Stockholm, Sweden) with 10% fetal bovine serum (FBS; Sigma-Aldrich, Darmstadt, Germany), gentamicin (20 µg/ml; Thermofisher), glutamine (2 mM; Thermofisher), and HEPES (0.01 M; Thermofisher), defined as complete medium (CM). The murine DC cell line JAWSII (ATCC CRL-11904) and neuroectodermal cell lines NE4Cs (ATCC CRL-2925) were cultured in DMEM supplemented with 10% FBS, gentamicin, glutamine, HEPES and human neuronal cell line SH-SY5Y (ATCC CRL-2266) was cultured in Opti-MEM supplemented with 10% FBS and gentamicin. All cultures were regularly tested for mycoplasma.

Bone marrow derived macrophages were differentiated in DMEM (Corning) with 10% FBS (Biowest) supplemented with 1% glutamine (Corning), 1% HEPES (Corning) and 10% CMG14 (Takeshita et al., 2000 (link)) supernatant for 7 days. 3T12 cells (ATCC, CCL-164, mycoplasma tested) were maintained in DMEM with 5% FBS supplemented with 1% glutamine and 1% HEPES. 293 T cells (ATCC, CRL-3216, mycoplasma tested), fibroblasts overexpressing human STING were maintained in DMEM with 10% FBS. Primary fibroblasts (MEFs) were isolated from embryonic tissue in DMEM with 10% FBS supplemented with 1% glutamine, 1% HEPES, then passed and maintained in the same culture medium for further propagation.

Three EAC cell lines were used in this study. SK-GT-4 cell line (DMSZ, Braunschweig, Germany) was originally isolated from an adenocarcinoma of the distal esophagus. OE33 cell line (ECACC, Salisbury, UK), established from an adenocarcinoma of the lower esophagus arising in BE and OACM5.1C cells, established from a lymph node metastasis derived from a primary adenocarcinoma of distal esophagus with the presence of BE were both purchased from ECCAC (Salisbury, UK). EAC cells were cultured in RPMI-1640 supplemented with antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B) and 10% FBS. A non-dysplastic BE derived cell line CP-A (ATCC, Teddington, USA) was used as a control to evaluate whether the effects of esomeprazole were specific of tumor cells. CP-A cells were cultured in MCDB-153 medium supplemented with 0.4 μg/L hydrocortisone (Sigma), 4 mM glutamine (ATCC), 20 mg/mL adenine (Sigma-Aldrich), 0.1 pM cholera toxin (Sigma-Aldrich), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium (Sigma), 150 μg/mL BPE (Sciencell), 20 ng/mL EGF (Sciencell), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B, and 5% FBS, as previously described (Peréz-Sayáns et al., 2010 (link)).