L glutamine

Mouse cell lines were generated from lung nodules of KRAS^G12D/LKB1^wt (K) and KRAS^G12D/LKB1^del (KL) transgenic mice as described in [34 (link)], and cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 2 mM of L-glutamine (Microgem) and 10% (v/v) fetal bovine serum (FBS, Euroclone). Cells were grown at 37°C in a humidified atmosphere supplemented with 5% (v/v) CO₂.
For cytotoxicity experiments, K and KL cell lines were seeded at respectively 6500 c/mL and 7000 c/mL, in 96-well plates and treated after 24 h with 2 mM of metformin (1,1-Dimethylbiguanide hydrochloride, #D150959 Sigma-Aldrich) and a 50% reduction of glucose, glutamine and FBS in the medium. Cell viability was examined after 72 h with MTS assay (Promega) and absorbance was acquired using a plate reader (GloMax Discover, Promega). The mean and SD of at least three independent experiments, each consisting of six replicates, are presented.
The cell lines are routinely tested by polymerase chain reaction for mycoplasma contamination.

The HMCLs used in this study, RPMI8226 (ATCC^® CCL-155TM) and OPM2 (ACC-50), were cultured in RPMI1640 medium (Lonza, Swiss) supplemented with 10% fetal bovine serum (FBS) (Euroclone S.p.A., Italy), 100 U/mL P/S (penicillin/streptomycin) (Microgem, IT, USA) and 2 mM L-glutamine (Microgem, Italy). Cell lines were seeded at 3 × 10⁵ cells/mL and split every 48 h. HPAECs (Human Pulmonary Artery endothelial cells—ATCC^® PCS-100-022TM) were cultured in Vascular Basal medium (ATCC^® PCS-100-030TM) supplemented with the Endothelial Cells Growth Kit-VEGF (ATCC^® PCS-100-041TM), following the manufacturer’s instruction (complete vascular medium). The HPAECs were seeded at a final concentration of 3 × 10³ cells/cm². The human BMSC line HS5 (ATCC^® CRL-11882™) was cultured in DMEM (Lonza, Swiss) supplemented with 10% FBS, 100 U/mL P/S and 2 mM L-glutamine.

Oreoch-1 cytotoxicity was tested using a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) assay on human colorectal adenocarcinoma (Caco-2) cells, purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). In detail, cells (3.5 × 10⁴/well), routinely grown in Dulbecco’s Modified Eagle Medium (DMEM) and supplemented with 1% antibiotic solution, 1% L-Glutamine, and 10% Fetal Bovine Serum (FBS) (Microgem, Pozzuoli, Italy), were seeded in a 96-multiwell plate and incubated at 37 °C in a humified atmosphere with 5% CO₂. The following day, the adherent cell monolayer was treated with decreasing peptide concentrations (200–1.56 μM), and resuspended in water. The untreated cells and cells exposed to 100% dimethyl sulfoxide (DMSO) were considered as negative (CTR−) and positive (CTR+) controls, respectively. After 20 h (h) of treatment, the cell viability was determined using a 5 mg/mL MTT solution (Sigma-Aldrich, St. Louis, MO, USA), by dispensing 100 μL in each well. After 3 h of incubation at 37 °C, the formazan crystals were dissolved with pure DMSO; then, the absorbance was read at 570 nm using a microplate reader (Tecan, Männedorf, Switzerland). In the end, the percentage of cytotoxicity was calculated according to the following formula: