L glutathione reduced

For all glutamine-free conditions, differentiation media (as described above) was made with DMEM/F-12 w/o glutamine and in the absence of 1% Glutamax (note: trace/negligible amounts of glutamine may be present in Matrigel). To achieve glutamine-starvation conditions, glutamine-free media was supplemented with 1 mM L-methionine sulfoximine (Millipore Sigma, 91016). Metabolite rescue experiments were performed under glutamine-starvation media in the presence of 8 mM dimethyl 2-oxoglutarate (Millipore Sigma, 349631), 60 μL/mL Embryomax nucleosides (Millipore Sigma, ES-008), 10 mM D-glucosamine 6-phosphate (Millipore Sigma, G5509), 2 mM D-(+)-glucosamine hydrochloride (Sigma-Aldrich, G1514), or 1 mM L-glutathione reduced (Millipore Sigma, G6529). Gln utilization was blocked by addition of 50 μM 6-diazo-5-oxo-L-norleucine (Millipore Sigma, D2141) or 1 μM CB-839 (Selleckchem, S7655) to either Gln-supplemented or Gln-free media. Gln, Arg, or Leu starvation/deprivation timed pulse experiments were performed by culturing cells in respective deprivation conditions for an initial 14 h or 24 h, washed with PBS, and then grown in Gln, Arg, or Leu replete conditions until D5 of differentiation.

Materials and Reagents. Silver nitrate, APTES, poly(sodium 4-styrenesulfonate) solution (average M_w ~ 70 000, 30 wt % in H₂O), poly(allylamine hydrochloride) (average M_w 50 000), 5,5′-ditho-bis (2-nitrobenzoic acid), β-nicotinamide adenine dinucleotide phosphate reduced, yeast glutathione reductase, L-glutathione reduced, L-cysteine, L-homocysteine, L-arginine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tyrosine, and L-valine were purchased from Sigma-Aldrich. P-type wafer was purchased from Silicon Quest (Santa Clara, CA). Isopropyl alcohol (IPA) and phosphate buffered saline were purchased from Fisher Scientific. All chemicals are reagent grade, and deionized water was obtained from a Milli-Q ultrapure (18.2 MΩ cm) system.

Erastin, RSL3, ML162, ML210, ferrostatin-1, liproxstatin-1, JAK inhibitor I and ruxolitinib were purchased from Cayman Chemical. Doxorubicin (Adriamycin) HCl and gemcitabine were purchased from Selleckchem. Deferoxamine mesylate salt, L-Glutathione reduced, sulfasalazine, L-Buthionine-sulfoximine, 2-Mercaptoethanol, and SIINFEKL peptide (OVA 257–264) were purchased from Sigma-Aldrich. Recombinant human IFNγ (285-IF) and mouse IFNγ (485-MI) were purchased from R&D. BODIPY 581/591 C11, anti-IFNγ (XMG1.2) and anti-TNFα (MP6-XT22) blocking antibodies were purchased from Thermo Fisher Scientific. Liperfluo was purchased from Dojindo Molecular Technologies. Cyst(e)inase was obtained from the laboratory of Everett Stone and George Georgiou (University of Texas at Austin, TX, USA).