Mao a

The chemical reagents of α-glucosidase from Saccharomyces cerevisiae, p-nitrophenyl glucopyranoside (pNPG), lipase type II from porcine pancreas, p-nitrophenyl butyrate (pNPB), vanillic acid, 4-aminoantipyrine, horseradish peroxidase, tyramine, MAO-A, galantamine, acetylthiocholine iodide (ATCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), Tris, acetylcholinesterase (AChE), levodopa (L-DOPA), and tyrosinase were acquired through Sigma-Aldrich (Madrid, Spain); clorgyline and α-kojic acid were from Cymit quimica (Barcelona, Spain); MgCl₂·6H₂O, HCl, NaCl, and potassium phosphate were from Panreac (Barcelona, Spain).

The potential involvement of creatine and guanidinoacetate in H₂O₂ production via MAO-A (Sigma Aldrich Poole, United Kingdom) was evaluated using Amplex^® red reagent hydrogen peroxide/peroxidase fluorescence assay kit (Invitrogen, Paisley, United Kingdom). It was previously shown that the use of MAO-A substrate tyramine allows evaluation of H₂O₂ production in substrate concentration dependant manner (Mazzio and Soliman, 2004 (link)) and this effect is irreversibly inhibited by phenelzine (Riederer et al., 2004 (link); Youdim et al., 2006 (link); Di Lisa et al., 2009 (link)).

The following chemical reagents: gallic acid, xanthine, NBT (nitroblue tetrazolium), xanthine oxidase, DPPH (2,2 Diphenyl 1 picrylhydrazyl), galantamine, ATCI (acetylthiocholine iodide), DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)), Tris, vanillic acid, 4-aminoantipyrine, horseradish peroxidase, acetylcholinesterase, tyramine, MAO-A, L-DOPA (levodopa) and tyrosinase were obtained through Sigma-Aldrich (Madrid, Spain). Clorgyline and α-Kojic acid were sourced from Cymit quimica (Barcelona, Spain). Na₂CO₃, HCl, NaCl, Methanol and potassium phosphate were acquired from Panreac (Barcelona, Spain). Juglone (5-hydroxy-1,4-naphthoquinone) and FUdR (5-fluoro-2′-deoxyuridine) were sourced from Alfa Aesar (Ward Hill, MA, USA). C. elegans strains and Escherichia coli OP50 were obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN, USA).

IC₅₀ values of LSD1 demethylase inhibition were obtained by the peroxidase-coupled reaction method as described previously [35 (link), 36 (link)]. Briefly, 1 μM human LSD1 was incubated with serial dilutions of inhibitors in 50 mM HEPES-Na (pH 7.5) buffer containing 400 μM 4-aminoantipyrine, modified Trinder’s reagent TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt dihydrate), and 40 μg/ml horseradish peroxidase at 25°C for 10 min. The reaction mixture was subsequently incubated with 100 μM K4-dimethylated H3 tail peptide (1–20) for 30 min. Absorption of the peroxidase by-product generated by lysine demethylation was measured at 562 nm with a 96-well microplate reader (Ultrospec Visible Plate Reader II 96; GE Healthcare). IC₅₀ values were calculated with Prism 6 software (version 6.0e), using dose-response results in triplicate. LSD2 inhibition assays were performed using 200 μM K4-dimethylated H3 tail peptide (1–20) and 1 μM human LSD2. MAO inhibition assays were performed using 50 or 150 μM tyramine, 100 μg/ml MAO-A (Sigma-Aldrich) and 200 μg/ml MAO-B (Sigma-Aldrich).