br In cell viability experiments performed using the tetrazo
In cell viability experiments performed using the tetrazolium dye MTT, experiments were performed in 96-well tissue culture plates. Briefly, 4 × 103 A-769662 were allowed to seed overnight in a 37 °C in-cubator + 5% CO2. The next day, cells were treated as indicated, then assessed for viability 24 h post-treatment. Cell culture media was as-pirated from cells, then replaced with 0.5 mg/mL MTT dissolved in fresh 1× DMEM and allowed to incubate for 2 h in a 37 °C in-cubator + 5% CO2. Number of viable cells was assessed by quantifying the amount of MTT reduced to insoluble formazan. Insoluble formazan was solubilized in 10% SDS + 0.01 N HCl. The absorbance at 570 nm, with reference wavelength at 690 nm, was measured. Data are pre-sented as average % change ± SEM from control(s) of n = 3 in-dependent experiments.
2.5. Western blot analysis
Whole cell lysates were prepared after treatment in RIPA lysis buffer containing 150 mM NaCl, 5 mM EDTA pH 8.0, 50 mM Tris pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS and 1× protease inhibitor cocktail. Soluble lysates were quantified using a Pierce™ BCA Protein Assay Kit (23225) from ThermoFisher Scientific (Waltham, MA), 20 μg cell lysates from samples were separated on 4–12% SDS-PAGE gel and transferred to poly(vinylidene difluoride) (PVDF) mem-brane. Membranes were blocked with 5% nonfat dried milk and in-cubated for 1 h room temperature or 4 °C overnight. Primary (1:1000 dilution) and horseradish peroxidase (HRP)-conjugated secondary (1:10,000 dilution) antibody incubations were performed at room temperature for 1 h. Immunofluorescent signals were detected with Pierce ECL Plus Western blotting substrate (32132) from ThermoFisher Scientific.
2.6. High performance liquid chromatography-mass spectrometry
Glutathione (GSH), γ-Glu-Cys, cysteine and cystathionine (CTH) were measured utilizing high pressure, liquid chromatography-mass spectrometry (HPLC-MS) as previously described . ~5 × 106 cells were lysed via three probe sonication in 200 μL of 10 mM N-ethylma-leimide (NEM) + 10 mM ammonium acetate, pH 7.4. To the lysate, 800 μL of methanol was added and the samples were vortexed. Samples were then centrifuged at 16,000 ×g for 5 min, then supernatants were collected into microcentrifuge tubes and dried via vacuum centrifuga-tion. Samples were further dried with 100 μL methanol, then 100 μL benzene. The carboxy termini of metabolites were esterified with the treatment of 100 μL of 3 N methanolic HCl for 60 min, 60 °C. Samples were then dried via vacuum centrifugation, redissolved in 50 μL of water and transferred to liquid chromatography injector vials. Kinetex XB-C18, 100 × 2.1 mm, 1.7-μm particle size, 100 Å pore diameter, re- verse phase column from Phenomenex (Torrance, CA) column was equilibrated with 85% 0.1 mM perfluorooctanoic acid in water (eulant
2.7. D4-cystine uptake assay and H2S donation
1× DMEM with high glucose but no glutamine, methionine, and cystine (D0422) was procured from Sigma Aldrich. This media was then supplemented with 3.97 mM glutamine, 200 μM methionine, 10% FBS and 200 μM D4-CC (DLM-1000-PK), which was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). ~1 × 106 cells were seeded in 100 mm tissue culture dishes and allowed to grow for 48 h at 37 °C + 5% CO2. In experiments where H2S-donor GYY 4137 was used, either 40 μM GYY 4137 or DMSO vehicle control was added for 48 h. Cells were harvested and processed in the identical manner described above (Section 2.6). Reported D4-CC uptake was measured by quanti-fying intracellular D2-cysteine levels normalized to total μg protein. Data are presented as average fold-difference from control or compar-ison group ± SEM of n = 3 independent experiments.
2.8. Nuclear fraction enrichment
5 × 106 cells were scraped from 100 mm tissue culture dishes on ice and washed with cold, 1× PBS (phosphate buffer saline). Cells were suspended in cold lysis buffer containing the following: 10 mM HEPES pH 7.7, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% NP-40, 0.5 mM PMSF and protease inhibitor cocktail. Cells were in-cubated on ice for 15 min with intermittent mixing, then centrifuged at 12,000 ×g at 4 °C for 10 min. The supernatant was collected as the cytosolic enriched fraction. The nuclear pellet was washed three times with cold lysis buffer, then resuspended in cold nuclear extraction buffer containing 20 mM HEPES pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail. After incuba-tion on ice for 30 min, the enriched nuclear fraction was collected by centrifugation at 12,000 ×g at 4 °C for 15 min.