L glutamine

Firefly luciferase-expressing CT-26 mouse colon carcinoma cells (luc-CT26, CSC-RR0237, Creative Biogene, Shirley, NY, USA) were grown at 37°C in a 5% CO₂ humidified atmosphere in Dulbecco’s Modified Eagle’s Medium (D6429, Dominique Dutscher, Brumath, France) supplemented with 10% foetal bovine serum (500105N1DD, Dominique Dutscher), 0.2% glucose (19002–013, Gibco, Thermo Fisher Scientific, Hampton, NH, USA), 2 mM L-glutamine (X0550, Dominique Dutscher), 100 U/ml penicillin and 100 μg/ml streptomycin (15140155, Gibco, Thermo Fisher Scientific). Cell suspensions were prepared by enzymatic treatment with trypsin–EDTA (11560626, Thermo Fisher Scientific). After centrifugation, 30 000 cells were suspended in a 200 μl injection volume of 0.9% saline solution for further orthotopic grafts.

Porcine pepsin (EC number 3.4.23.1, from porcine gastric mucosa, >250 U mg⁻¹), pancreatin from porcine pancreas (4× USP specifications, European commission number: 232-468-9), diprotin A, Gly-Pro-7-amido-4-methylcoumarin hydrobromide (H-Gly-Pro-AMC, HBr) were purchased from Sigma-Aldrich (Villefranche-sur-Saône, France). Dulbecco’s modified Eagle’s cell culture Medium (DMEM), trypsin, L-glutamine, fetal bovine serum, antibiotics (penicillin and streptomycin) were purchased from Dutscher (Issy-les-Moulineaux, France). Fish byproduct protein hydrolysate (FBPH) was obtained by the hydrolysis of tilapia (Oreochromis niloticus) byproducts (FBP, bones and viscera), using commercially available food grade enzymes. FBPH and FBP were provided by Diana Pet Food (Elven, France). Synthetic peptides were purchased from GeneCust (Boynes, France).

Human U87-MG glioblastoma cells (ATCC HTB-14) were modified by lentiviral infection to stably express fLuc2 and eGFP (U87-fLuc2/eGFP, maximum excitation λ = 488 nm, maximum emission λ = 509 nm) under the control of the CMV promoter (kindly provided by Dr. S.A. Collins from Department of Medicine-Digestive Diseases, UCLA, USA)48 (link). They were grown at 37 °C in a 5% CO₂ humidified atmosphere, in Modified Essential Medium EARLES (10370–047, Gibco, France) supplemented with 10% fetal bovine serum (10500–064, Gibco, France), 0.2% Glucose (19002–013, Gibco, France), 2 mM L-glutamine (X0550, Dominique Dutscher, France) and 100 U/ml penicillin and 100 μg/ml streptomycin (15140155, Gibco, France). The viability of U87 cells was greater than 90% as determined by visual counts using Trypan Blue Dye Exclusion on a Malassez cell, or by analysis with the Muse^® Cell Analyzer (MCH100102, Muse Count & Viability Assay Kit, Millipore).

The LS174T, a well-differentiated human colonic goblet cell line [19 (link)], was purchased from Sigma-Aldrich (product number 87060401-1VL Saint Quentin Fallavier, France). Cells were routinely grown as a monolayer in 75 cm² plastic flasks and cultured at 37 °C in a humid environment containing 5% CO₂ in MEM medium supplemented with 2 mM L-Glutamine (Dutscher, Brumath, France); 10% heat-inactivated fetal bovine serum (FBS); 1% non-essential amino acids (Dutsche, Brumath, France); and an antibiotics cocktail containing 100 U/mL penicillin, and 100 mg/mL streptomycin and amphotericin B (Dutscher, Brumath, France). Cells were seeded in six-well plates with 4 × 10⁵ cells/well. When cells reached 80% confluence, they were treated with different stimuli. PAL, DHA, EPA, LNA, OA, AA, and LA were dissolved in 100% ethanol at a 1 mM concentration, flushed with nitrogen, and stored at −20 °C until use. Before treatment, fatty acids were placed at room temperature, except for PAL, which was heated in a 40 °C water bath. Fatty acids were added to 1 mL of MEM containing FA-free BSA to be combined at a 4:1 ratio. The specific treatment concentrations and incubation times are shown in the figure legends. For the control, cells were treated with the same quantity of ethanol and MEM-BSA concentrations as cells treated with FA.

Human recombinant TGF-β1 was obtained from PeproTech, (Cranbury, NJ, USA). FICZ and kynurenine were purchased from Sigma (St. Louis, MO, USA). ITE and curcumin were purchased from TOCRIS (Bristol, UK). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, USA) and penicillin, streptomycin, L-glutamine from Dutscher (Brumath, France). MEM non-essential amino acid and Cellytic™ buffer were supplied by Sigma (St. Louis, MO, USA). Minimum Essential Medium Eagle was obtained from Eurobio (Montpellier, France).

For the in vitro testing of the efficacy of the molecules, the P. falciparum artemisinin-resistant strain F32-ART and its twin artemisinin-sensitive line F32-TEM, selected by Witkowski et al. [34 (link)], were used. The parasites were in RPMI 1640 with 5% serum and 2% hematocrit at 37 °C in a humidified 5% CO₂ atmosphere [35 (link)]. For each experiment, the parasites were tightly synchronized by 5% D-sorbitol treatment at the ring stage (0–24 h) [36 (link)].
Vero cells, a non-cancerous mammalian cell line from the kidney of an African green monkey (Cercopithecus aethiops), were used for a safety assessment and subsequent determination of the selectivity indices of the molecules. The Vero cells were cultured in MEM (Dutscher, France) supplemented with 10% fetal bovine serum (Dutscher), 1X non-essential amino acids (Dutscher) 100 U/mL, 100 µg/mL penicillin/streptomycin (Dutscher), and 2 mM L-glutamine (Dutscher) at 37 °C in a humidified 5% CO₂ atmosphere [37 (link)].

Human epithelial breast cancer cell lines, MDA-MB-231 (ATCC® HTB-126) and MDA-MB-468 (ATCC® HTB-132™), were purchased from American Type Culture Collection (ATCC, Manassas, USA) and cultured in Eagle’s MEM medium (Gibco®) supplemented with 1 mM l-glutamine, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 4 μg/mL gentamycin and 10% fetal calf serum (Dutscher, Brumath, France). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.