Cystine

Cell culture experiments were performed with THP-1 monocytes (ATCC®, Manassas, VA), MM.1S B lymphoblasts (ATCC®) and primary PBMCs from healthy donors (AllCells, Alameda, CA). Reduced and oxidized conditions were created by adding 300 μL of 10 mM cysteine and 150 μL of 10 mM cystine (Sigma-Aldrich, St. Louis, MO), respectively, to 15 mL DMEM/F12 media without methionione, cysteine, cystine and glutamine (HyClone, Life Technologies). Media was supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Cellgro, Corning Life Sciences, Corning, NY), and 50 mg/mL gentamicin sulfate (Cellgro). Cells were re-suspended to a concentration of 1 million cells/mL and cultured for 4 hours at 37°C with 5% CO₂. In selected experiments, cells were also exposed to 100 nM dexamethasone for 1 hour (Sigma-Aldrich), added at hour 3 of incubation.

The 13 primary GBM cell lines used in this study were isolated from GBM patient tissues and authenticated by neuro-pathologist at The Ohio State University (OSU34, OSU35, OSU38, OSU53, OSU61, OSU68, OSU94, OSU96, MDNSC2, MDNSC20, MHG8, MGH74, and 146). The 4 commercially available cell lines (U118, U87, LN18, and LN229) were obtained from ATCC. GBM cells were maintained in DMEM (Life Technologies), supplemented with 10% FBS (Sigma-Aldrich), and 1% antibiotic-antimycotic (Life Technologies). Normal Human Astrocytes were obtained from Lonza and were maintained in Clonetics™ AGM™ Astrocyte Growth Medium (Lonza). Cells were cultured at 37° C under a gas phase of 95% air and 5% CO₂. All studies were conducted within 5 passages. Before extracting intracellular and extracellular metabolites from Normal Human Astrocytes for metabolic profiling, the cells were grown in the same DMEM media as the GBM cell lines to maintain dietary consistency. In order to have a methionine free-DMEM for the methionine dependence studies, we used DMEM absent of glutamine, methionine, cystine, and pyruvate (Life Technologies) and supplemented with glutamine (4mM), cystine (0.2mM), and pyruvate (1mM). Methionine (20 μM) was added to the methionine free-DMEM for low methionine studies.

Cells were grown to ~80% confluency at the time of assay. Complete medium was replaced by AHA medium formulated with DMEM, high glucose, no glutamine, no methionine, no cystine (Gibco), 10% dialyzed FBS (Gibco), 1% GlutaMAX (Gibco), 1% Penicillin-Streptomycin (Gibco), 62.6 mg/L L-Cysteine (Sigma), 250uM L-Azidohomoalanine (AHA, Click Chemistry Tools). For non-AHA control, the AHA was replaced by 30 mg/L L-methionine (Sigma). For Anisomycin (ANS) treatment, AHA medium was further added with 10 μg/mL Anisomycin (Cell Signaling). After 3 h of incubation, cells were scraped in cold DPBS, washed twice with cold DPBS and lysed in RIPA buffer. Protein concentration of the lysate was quantified by BCA and input protein concentrations were normalized to 2.1 mg/mL for downstream click chemistry. 50μL of the normalized cell lysate was used for click reaction with biotin-Alkyne (Click Chemistry Tools) by using Click-&-Go Click Chemistry Reaction Buffer Kit (Click Chemistry Tools). The reaction mixture was then methanol precipitated and was resuspended in 2xLaemmi buffer (BIO-RAD) with 2-mercaptoethanol (BIO-RAD) and denatured at 95°C for 10 min. Final protein concentration is 1 mg/mL. The sample was then resolved by SDS–PAGE, and total protein signal was detected by One-Step Blue Protein Gel Stain (Biotium). The biotin signal was detected by HRP-Conjugated Streptavidin (Thermo Scientific).