Nadph

Immediately after treatment with auranofin, cells were washed twice with PBS (1×) on ice then treated with 100 µL of 1% Triton x-100 supplemented with 1x HALT protease cocktail (ThermoFisher Scientific) to lyse the cells. The lysed cells were then re-pelleted at 12,000 g for 10 min. The supernatant of the solutions was then collected and the protein content in the solution was assessed via the bicinchoninic acid (BCA) assay. 40 μg of protein were then added to a solution containing 400 μM NADPH (VWR) and 800 μM selenocystine (ThermoFisher Scientific). The absorbance of the solution at 340 nm was then measured every 2 min for a period of 90 min.

NaCl, D-glucose, KH₂PO₄, NaF, dihydroethidium (DHE, stock 10 mM in DMSO), Tris-base, Dulbecco's Modified Eagle Media (DMEM), fetal calf serum (FCS), penicillin/streptomycin, 1x minimal essential amino acids, and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF, stock 10 mM in DMSO) were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Acetylcholine, phenylephrine, leupeptin, aprotinin, sodium orthovanadate, pepstatin, phenylmethylsulfonyl fluoride (PMSF), NaHCO₃, ethylene glycol tetraacetic acid (EGTA), A23187 (calcium ionophore, stock 10 mM in DMSO), sodium deoxycholate, NP40, lucigenin, diethylenetriaminepentaacetic acid (DTPA), apocynin, and endothelial cell growth supplement were all purchased from Sigma-Aldrich (St. Louis, MO, USA). KCl, CaCl₂, MgSO₄, and ethylene-diaminetetraacetic acid (EDTA) were all purchased from EMD Millipore (Billerica, MA, USA). Sodium dodecyl sulfate (SDS) and NADPH were purchased from VWR (Radnor, PA, USA). Alexa-647-labeled Annexin V were purchased from BioLegend (San Diego, CA, USA). Heparin was purchased through Leo Pharma (Thornhill, ON, Canada). A complete list of all antibodies used and their sources is provided in Table 1.

Mifepristone and misoprostol (neat oil) were purchased from Cayman Chemical (Ann Arbor, MI, United States). Urethane, Tween^® 80, 5,5′-dithiobis(2-nitrobenzoic acid), 2-thiobarbituric acid (TBA), L-glutathione reduced (GSH) and glutathione reductase from baker’s yeast were purchased from Sigma-Aldrich (St. Louis, MO, United States). Carboxymethylcellulose sodium (CMC-Na), 2-vinylpyridine, NADPH, 1-chloro-2,4-dinitrobenzene and trichloroacetic acid (TCA) were purchased from VWR (Philadelphia, PA, United States). All other chemicals were of the highest grade commercially available.