Glutathione ethyl ester

Intracellular glutathione levels were measured [14 (link)]. Briefly, 1 × 10⁶ PBMCs were resuspended in 1 mL of a 50 mM monochlorobimane (Sigma, St. Louis, MO, USA) solution, and incubated for 15 min at 37 °C in the dark. After incubation, the stain was quenched by adding 50 µL of trichloroacetic acid to each sample and samples were centrifuged at 10,000 rpm for 5 min at room temperature. One milliliter of the supernatant was then layered on top of an equal volume of dichloromethane in a glass tube and the sample was centrifuged at 3500 rpm for 2 min at room temperature. The aqueous supernatant (200 uL/well) was plated in duplicate in an opaque 96-well plate and the fluorescence was read immediately on a Spectra GemniEM plate reader at excitation 360/40 nm and emission 460/40 nm. Standards of known concentrations of reduced glutathione were prepared in parallel with each sample and were used to calculate glutathione concentration. Jurkat cells (1 × 10⁶) were treated with 2 mM glutathione ethylester (Sigma, St. Louis, MO, USA) for 15 min or 2 mM hydrogen peroxide (Sigma, St. Louis, MO, USA) as a positive control and negative control, respectively. Results were analyzed using Microsoft Excel 2010. Mean fluorescence for each sample was calculated by subtracting baseline fluorescence (unstained cells) then normalizing it to the cell number.

BioGEE was purchased from Molecular Probes (Eugene, OR, USA) or synthesized from 25 mM EZ-Link-Sulfo-NHS-Biotin (Thermo Fisher Scientific, Nepean, ON, Canada) and 25 mM glutathione ethyl ester (Sigma Chemical Co., St Louis, MO, USA) in 50 mM phosphate buffered saline at pH 8. After 1 h incubation, NH₄HCO₃ was added to a final concentration of 138 mM to quench the remaining EZ-Link-Sulfo-NHS-Biotin. The concentration of the newly synthesized BioGEE was determined spectrophotometrically with 5,5′-dithiobis-(2-nitrobenzoic acid) using GSH as standard. Except when indicated otherwise, buffers, chemicals, and reagents were of analytical grade from Sigma Chemical Co. or Thermo Fisher Scientific.

BGM, HeLa, Vero, RD and MRC-5 cells were maintained in MEM (Gibco), supplemented with 10% fetal bovine serum (FBS) (Integro), 1% bicarbonate (Gibco) and 1% L-glutamine (Gibco). The synthesis of TP219 is described elsewhere [29] (link). Enviroxime was synthesized by Dr. G. Pürstinger (Institut für Pharmazie, Universität Innsbruck, Austria). Geldanamycin was from Biovision (Milpitas). L-Buthionine sulfoximine (BSO), guanidine hydrochloride (GuaHCl), N-acetyl cysteine (NAC), glutathione (GSH), glutathione ethyl ester (GEE) and dithiothreitol (DTT) were purchased from Sigma Aldrich.

N-acetyl-L-cysteine, L-aspartic acid, L-cysteine, dimethyl α-ketoglutarate (AKG), glutathione ethyl ester, L-homocystine, L-methionine, sodium α-ketobutyrate (AKB), and sodium pyruvate were purchased from Sigma. U-¹³C₃¹⁵N-L-cysteine was purchased from Cambridge Isotope Laboratories.

Anti-Bcl-xL (sc-634, 1:700), anti-p53 (sc-126, 1:1000), anti-p21 (sc-6246, 1:700), anti-Mcl-1 (sc-819, 1:1000), and anti-c-FLIP (sc-8347, 1:700) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-β-actin (A5441, 1:2000) antibody and glutathione ethyl ester (GEE) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti- poly (ADP-ribose) polymerase (PARP) (#9542, 1:1000) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-caspase-3 (610322, 1:1000) antibody was purchased from BD Biosciences (Bedford, MA, USA). Benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was purchased from R&D Systems (Minneapolis, MN, USA). PD-98059 (a MEK inhibitor; PD), SP600125 (a JNK inhibitor; SP), and SB-203580 (a p38 MAP Kinase inhibitor; SB) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). N-acetylcysteine (NAC) was purchased from Calbiochem (San Diego, CA, USA).