Glutamine

THP-1 cells were cultured in RPMI1640 (HyClone laboratories, Logan, UT) with 10% FBS (Atlantic biologicals, Miami, FL), 2mM Glutamine(Corning Cellgro, Manassas, VA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B(Corning Cellgro, Manassas, VA). Murine erythroleukemia (MEL), and 293T cells were maintained in DMEM High Glucose (HyClone laboratories, Logan, UT) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B. During the differentiation, MEL cell was induced by 5mM hexamethylene bisacetamide (HMBA), and supplemented with 20%FBS. Lineage negative cells purified using mouse lineage cell depletion kit (Miltenyi Biotechnology, San Diego, CA) were cultured in the Stem Span SFEM medium (Stemcell, Vancouver, Canada) with 100ng/ml mouse stem cell factor (SCF), 100ng/ml Flt3-Ligand, 100ng/ml IL3, and 100ng/ml TPO (Sigma, St Louis, MO).

The SJ-GBM2 cell line was obtained from the Children’s Oncology
Group. The GBM10 and GBM43 xenografts were a kind gift from Dr. Jann Sarkaria
(Mayo Clinic), and the tumors were expanded by passage in the flank of NOD/SCID
γ^null mice. To generate GBM10 and GBM43
cell lines, tumors were harvested, disaggregated, and maintained in 2.5% FBS for
14 days on Matrigel-coated plates (BD Biosciences) to remove murine fibroblasts,
as previously described.^{50 (link)}Cell lines were propagated in DMEM with 10% FBS medium with glutamine (Cellgro,
Manassas, VA) for no more than 7 passages in 5% CO₂ at 37 °C.
Cell line identity was confirmed by DNA fingerprint analysis (IDEXX BioResearch)
for species and baseline short-tandem repeat analysis testing. The pancreatic
cancer cells used in these studies were low-passage patient-derived PDAC cell
lines (fewer than 35 passages from patient tumors), which were received from Dr.
Anirban Maitra (The Johns Hopkins University), and maintained as previously
described.^{55 (link),91 (link)} Pa02C, Panc10.05, and Panc198 were
cultured in DMEM medium and supplemented with 10% FBS in 5% CO₂ at 37
°C for fewer than 10 passages. Cell line identity was confirmed by DNA
fingerprint analysis (IDEXX BioResearch) for species and baseline short-tandem
repeat analysis testing and cells were routinely checked for mycoplasma.

Cells were grown at 37 °C under a humidified 5% CO₂ atmosphere, in a culture medium consisting of high-glucose DMEM (Caisson Labs) for A431, HEK293T, NIH-3T3 and HeLa cells or RPMI (Caisson Labs) for Ramos, HCC827, and A549 cells. The SW620 and MDA-MB-435S cells were grown in L-15 media in a humidified atmosphere with no CO₂ added. All media was supplemented with 10% FBS (Gemini), penicillin (50 IU/mL), streptomycin (50 μg/mL), and glutamine (2 mM) (Cellgro). For SILAC experiments, the culture medium was replaced with either SILAC DMEM or SILAC RPMI (Thermo) as appropriate, and the medium was supplemented with 10% dialyzed FBS, penicillin, streptomycin, and glutamine. For the isotopically heavy cell samples, 100 μg/mL of both [¹³C₆,¹⁵N₄]L-arginine-HCl and [¹³C₆,¹⁵N₂]L-lysine-HCl (Sigma-Aldrich) was also added to the culture medium. For the isotopically light cell samples, the culture medium was supplemented with 100 μg/mL of both L-arginine-HCl and L-lysine-HCl. Cells were passaged at least six times in isotope-containing medium before being used for ABPP-SILAC experiments.