Ebselen

PBS, HBSS, DMEM/F12 (Thermo Fisher Scientific, Inc.), UW solution (Viaspan^®; Bristol-Myers Squibb, Co.), ET-Kyoto solution (Otsuka Pharmaceutical Factory, Inc.), and Optisol GS™ (Bausch & Lomb, Rochester, NY) were prepared as candidates for a basal preservation medium. The following additives were also prepared: dextran 40 (0.2, 2, 20 g/L; Wako), chondroitin sulfate A (0.01, 0.1, 1%; Sigma-Aldrich, St. Louis, MO, USA), N-acetylcysteine (0.1, 1, 10 mM; Wako), allopurinol (0.1, 1, 10 mM; Tokyo Chemical Industry Co., Ltd), glutathione (0.3, 3, 30 mM; Sigma-Aldrich), adenosine (0.05, 0.5, 5 mM; Sigma-Aldrich), hyaluronic acid (0.01, 0.1, 0.5%; Calbiochem), dibutyryl cAMP (0.2, 2, 20 mM; Enzo Life Sciences), trehalose (12, 120, 1200 mM; Hayashibara), tert-butylhydroquinone (tBHQ; 0.1, 1, 10 μM; Sigma-Aldrich), oltipraz (1, 10, 100 μM; Sigma-Aldrich), and ebselen (1, 10, 100 μM; Sigma). Each of these additives was added to HBSS individually.

Mtb was grown to mid-log phase and diluted to an OD of 0.03 in 7H9 medium. Bacteria were then exposed to 1.5-fold serial dilution of antimicrobial compounds. Optical density was recorded after 14 days and normalized to the corresponding strains without drug treatment. Minimum inhibitory concentration is defined as the lowest concentration of a drug at which bacterial growth was inhibited at least 90%, as compared to the control containing no antimicrobial compounds. Ampicillin, auranofin, D-cycloserine, ebselen, ethambutol, faropenem, hydroxyurea, isoniazid, kanamycin, levofloxacin, meropenem, mitomycin C, moxifloxacin, piperacillin, rifampicin, streptomycin and vancomycin were purchased from Sigma Aldrich, St. Louis, MO. Moenomycin was from Santa Cruz Biotechnology. Bedaquilline was received as a gift from C. Barry.

2-phenyl-1, 2-benzisoselenazol-3(2H)-one, ebselen (Sigma-Aldrich), was dissolved in tissue culture-grade DMSO (Sigma-Aldrich, St. Louis, MO.) to a stock concentration of 10 mM and stored at −20°C protected from light.

To evaluate the effect of exogenous ONOO^-, P. infestans hyphae were sprayed with ONOO^- generator (50 μM 3-Morpholinosydnonimine, SIN-1, Calbiochem), which gradually decomposed to yield equimolar amounts of NO and O₂^-. Scavenger of ONOO^- (50 μM ebselen, Sigma) were used to estimate the effect of endogenous ONOO^- on protein Tyr nitration. Control cultures were treated with sterile water. After 24 h of hyphae incubation protein 3-nitrotyrosine assay was performed.

Ebselen, ferric ammonium citrate (FAC), and anti-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-DMT1 was obtained from EMD Millipore (Rahway, NJ, USA). Anti-mTORC1 and anti-p-mTORC1 were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-FPN1, anti-p53, and anti-p21 were purchased from Abcam (Cambridge, CB2 0AX, UK). Anti-FT-L and cell culture reagents were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). All chemicals used in this study were of analytical grade.

Human LDL, 4‐nitrophenyl phosphate, ebselen, mouse antibody for α‐tubulin, and phosphatase inhibitor cocktails 1 and 2 were from Sigma‐Aldrich. Rabbit antibodies for CD36, sEH and phospho‐threonine (p‐Thr), and goat antibodies for SR‐A and ABCG1 were from Santa Cruz Biotechnology. Mouse antibodies for ABCA1, rabbit antibodies for SR‐BI and F4/80 were from Abcam. 12‐(3‐adamantan‐1‐yl‐ureido)‐dodecanoic acid (AUDA), NBD cholesterol, oleic acid (OA) and TBARS assay kits were from Cayman Chemical. Cholesterol and triglyceride assay kits were from Randox. Peroxidase‐conjugated antimouse IgG, anti‐rabbit IgG, anti‐goat IgG and anti‐rat IgG antibodies were from Jackson ImmunoResearch.