L arginine

Cytotoxic T Lymphocytes were generated as previously described (Hukelmann et al., 2016 (link), Navarro et al., 2011 (link), Navarro et al., 2014 (link)) and expanded in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (Life Technologies), 50 units/mL penicillin-G, 50 μg/mL streptomycin, and 50 μM β-mercaptoethanol and in the presence of 20 ng/mL IL-2 alone (Proleukin, Novartis). For SILAC labeling, CTLs were cultured for 5 days in SILAC RPMI 1640 medium (Life Technologies), supplemented with 200 mg/L L-proline, 84 mg/L L-arginine, 300 mg/L L-glutamate, 10% dialyzed FBS with a 10 kDa cutoff (Thermo Scientific), 50 units/mL penicillin-G, 50 μg/mL streptomycin, 50 μM β-mercaptoethanol, and 20 ng/mL IL-2. The “light” SILAC media contained L-[12C6, 14N4]arginine (R0) and L-[12C6, 14N2]lysine (K0). The “heavy” media contained L-[13C6, 15N4]arginine (R10) and L-[13C6, 15N2]lysine (K8).
For the IL-2 stimulation of CTLs, cells were “IL-2 quiesced” by the removal of IL-2 for 24 hr, but they were supplemented with 20 ng/ml IL-12 (R&D Systems) to sustain cell viability (at ∼90%) and the expression of the IL-2Rα chain (CD25). For the Tofacitinib and PP2 studies, CTLs were maintained only in the presence of IL-2, and no IL-12 was added to the culture.

M and S cells were cultured in L-lysine and L-arginine-depleted RPMI (Thermo Scientific, Hudson, NH) supplemented with 10% dialyzed FBS, antibiotics (PAA), and either 0.1 mg/mL ¹²C₆- (M) or 0.1 mg/mL ¹³C₆- (S), L-lysine and L-arginine (Thermo). The medium was replaced every 2 days, and cells routinely passaged at 80-90% confluence. After 14 days, cells were cultured for 2 days in light (M) or heavy (S) labeled medium without FBS, conditioned mediums collected, centrifuged, filtered through a 0.2 μm filter (VWR) and concentrated using Amicon centrifugal filter devices with a 3-kDa molecular weight cut-off (Millipore, Billerica, MA). Protein content was quantified (Bradford RcDc protein assay; BioRad, Hercules, CA) and 15 μg of each sample were mixed, diluted in 50 mM ammonium bicarbonate and concentrated using an Amicon centrifugal filter device with a 10-kDa molecular weight cut-off (Millipore). The <10 kDa fraction was evaporated to dryness, resuspended in 8 M urea, 50 mM ammonium bicarbonate, reduced with 50 mM dithiothreitol, alkylated with 125 mM iodoacetamide, digested with trypsin and analysed by LC-MS. The >10 kDa fraction was resuspended in loading buffer and subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel.

HEK293T and MEF cells were grown in Dulbecco’s modified Eagle’s medium, with GlutaMAX^TM supplement (DMEM + GlutaMAX, Gibco) with 10% fetal bovine serum and 1% Penicillin–Streptomycin at 37 °C, with 5% CO₂. Then tRNA transfections were performed in 6-well plates. In vitro transcribed tRNA was mixed with the 4 µg of Arg sensor plasmid, and this mixture was directly added to the cells in combination with Lipofectamine 2000. For Figure 3, right, 1200 ng tRNA was transfected. For Figure 4, we used dialyzed serum (A33820-01, Gibco) and DMEM media deficient in both L-lysine and L-arginine (88364, Thermo Scientific). L-lysine was added to make the 146 mg/L final concentration. After 48 h, the transfected cells were washed once with phosphate-buffered saline (PBS, Corning) and harvested by scraping and centrifugation. Cell pellets were lysed in 4 × SDS Sample Buffer at the w:v ratio of 1:20 (1 mg cell pellet: 20 µL buffer), followed by boiling the samples for 10 min. Then 10 µL of each sample was loaded for SDS–PAGE electrophoresis.

Stably transfected hNgb-EGFP and EGFP control SH-SY5Y cells were grown in the complete growth medium. For SILAC (stable isotope labeling with amino acid in cell cultures) experiments, the hNgb-EGFP SH-SY5Y cells were maintained in DMEM:F12 (1:1) medium for SILAC (Thermo Fisher Scientific), supplemented with 2 mM L-glutamine (Gibco), 10% dialyzed, heat-inactivated fetal bovine serum (Sigma-Aldrich), and 100 U/ml penicillin-streptomycin (Gibco). L-arginine (Arg0) and L-lysine (Lys0) (‘Light’), ¹³C₆¹⁴N₄-L-arginine (Arg6) and 4,4,5,5-D4-L-lysine (Lys4) (‘Medium’), or ¹³C₆¹⁵N₄-L-arginine (Arg10) and ¹³C₆¹⁵N₂-L-lysine (Lys8) (‘Heavy’) were used for metabolic labeling (0.1 g/L, Thermo Fisher Scientific). In the “reverse” experiment, the labels were moved to the next condition in line as compared to the first run. For the analysis of hNgb-protein interactions under ferroptosis, 50 μM of erastin (Selleckchem) was added to the medium of the labeled cells for the last 24 h of culture.

Compounds JQ1, I-BET151, estradiol, 17-AAG, GW7604 and raloxifene were either available from the GSK compound collection or sourced commercially with purity of 98% or higher. Reagents and media were purchased from Sigma (St. Louis, MO) unless otherwise noted. SILAC “light” (L) medium for Jurkat and primary human T cells was made of basal SILAC RPMI medium supplemented with 10% dialysed fetal bovine serum (FBS), glucose (2 g/L), L-glutamine (2 mM), phenol red (all Thermo Fisher) as well as L-Arginine and L-Lysine (100 mg/L, each, Thermo Fisher). The corresponding SILAC “heavy” (H) medium contained stable isotope-labeled ¹³C₆¹⁵N₂-L-Lysine and ¹³C₆¹⁵N₄-L-Arginine (Thermo Fisher), both at 100 mg/L, instead of the “light” amino acids.