Aminoguanidine

Dieckol (DK) isolated from Ecklonia cava were kindly provided by Prof. You-Jin Jeon (Jeju Notional University, Republic of Korea) [11 (link)]. MGO and aminoguanidine (AG) were ordered from Sigma-Aldrich (St. Louis, MO, USA). Reagents used in all experiments were purchased in analytical grade. Information on the antibody used for Western blot is presented in Supplementary File (Table S1).

Aminoguanidine (AG), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), D-glucose, sodium azide, bovine serum albumin (BSA), and methylglyoxal (MGO) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Folin–Ciocalteu reagent, sodium carbonate, and ethanol (HPLC purity) were provided by POCh (Gliwice, Poland). Water was purified with the Mili-Q-system (Millipore, Bedford, Massachusetts, USA). PCL assay kit was purchased from Analytical Jena (Jena, Germany). To prepare the phosphate buffer (0.1 M, pH 7.4), monosodium phosphate and dipotassium phosphate were mixed at the appropriate ratio.

Bovine serum albumin (BSA), aminoguanidine (AG), methylglyoxal (MG), acetylcholine (ACh), phenylephrine (PE), Nω-Nitro-L-argininemethyl ester hydrochloride (L-NAME), and diphenyl-2-picrylhydrazyl (DPPH) were all purchased from Sigma–Aldrich, Dorset, UK. Ultrapure deionized water was used as solvent except for the natural compounds and DPPH, which were dissolved in dimethyl sulfoxide (DMSO) in a concentration that did not exceed 0.1% in the reaction media. All other solvents and chemicals used were of analytical grade.

The solvent used was ethanol supplied by Merck Millipore (Darmstadt, Germany), and the reagents trolox, gallic acid, 2,2-diphenyl−1-picrylhydrazyl (DPPH), acetonitrile, phosphoric acid, oleuropein, 3-hydroxytyrosol, aminoguanidine, D(+)-glucose, bovine serum albumin (defatted fraction), and sodium azide were supplied by Sigma Aldrich (St. Louis, MO, USA).

Cells were seeded with fresh medium with 2% serum for all drug treatments. Following overnight attachment, cells were treated and incubated at 37°C for times indicated below. Difluoromethylornithine (DFMO; TargetMol) was diluted to a 100-mM solution in sterile water and stored at 4°C. For DFMO, cells were treated and incubated for 96 h at 1 mM. N11-Diethylnorspermine (DENSpm; Santa Cruz Biotechnology) was diluted to 10 mM in sterile water and stored at 4°C. For DENSpm, cells were treated and incubated for 24 h at 100 mM. GC7 (Cayman Chemical) was diluted to a 100-mM solution in sterile water and stored at −20°C. For GC7, cells were treated for 24 h at 500 mM along with 500 μM aminoguanidine (Sigma-Aldrich). Ribavirin (VWR) was diluted to 100 mM in sterile water and stored at −20°C. For Ribavirin, cells were treated and incubated for 4 h at 400 mM. Polyamines (Sigma-Aldrich) were added to cells at the time of infection at 10 mM unless otherwise indicated. For infections, all drug treatments were performed at the time of infection unless otherwise indicated. For persistently infected experiments, drugs were continually supplemented onto cells with fresh medium with 2% serum upon collection of viral supernatants.