L glutamic acid monosodium salt monohydrate

The following ARs agonists and antagonists were purchased from Tocris (Bristol, UK): NECA (5′-(N-Ethyl-carboxamido)adenosine); CGS15943 (9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine); DPCPX (1,3-Dipropyl-8-cyclopentylxanthine); SCH442416 (2-(2-Furanyl)-7-[3-(4-methoxy-phenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine); PSB603 (8-(4-(4-(4-Chlorophenyl)piperazide-1-sulfonyl) phenyl)-1-propylxanthine). CCPA (2-Chloro-N6-cyclopentyladenosine) and MRS1523 (3-Propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate), adenosine deaminase (ADA), l-glutamic acid monosodium salt monohydrate, forskolin, KT5720, tert-Butyl hydroperoxide (TBHP), N-acetylcysteine (NAC) were obtained from Sigma-Aldrich (St. Luis, MO, USA). Annexin V AlexaFluor^TM 488 Ready Flow Conjugate, SYTOX™ AADvanced™ Dead Cell Stain, CellROX™ Green Reagent, MitoTracker™ Red CMXRos were purchased from Life Technologies (Monza, Italy). The A₁AR positive allosteric modulator TRR469 was previously synthesized and characterized (compound 4ad in [23 (link)]).

The yeast strain used in this study was Saccharomyces cerevisiae BY4741 (MATa, his3Δ1, leu2Δ0, met17Δ0, ura3Δ0) [26 (link)]. Cultivation was carried out in liquid or solid (20 g L⁻¹ w/v agar) YPD (10 g L⁻¹ w/v yeast extract, 20 g L⁻¹ w/v peptone, 20 g L⁻¹ w/v glucose). Selection medium was 1.7 g L⁻¹ yeast nitrogen base without amino acids and ammonium sulphate (Millipore), 1 g L⁻¹ L-glutamic acid monosodium salt monohydrate (Sigma-Aldrich), 20 g L⁻¹ w/v glucose, 120 mg L⁻¹ L-leucine (Sigma-Aldrich), 20 mg L⁻¹ uracil (Sigma-Aldrich), 200 µg ml⁻¹ Hygromycin B (Invivogen) and 20 g L⁻¹ w/v agar. All yeast cultivation was done at 30 °C.

The following reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA): L-glutamic acid monosodium salt monohydrate, N-acetylcysteine (NAC), Tween 20, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), aqueous mounting buffer, sodium hydroxide, sulfanilamide, N-(1-naphthyl)-ethylenediamine dihydrochloride, and phosphoric acid. Other reagents were obtained from the following manufacturers: Acetyl alcohol, paraformaldehyde, H₂O₂, citrate buffer, methylene alcohol and Triton X-100 were obtained from Duksan Science, Seoul, Korea; Daejung Chemicals & Metals Co., Siheung, Korea; and Junsei Chemical Co., Ltd., Tokyo, Japan. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), penicillin–streptomycin solution, and trypsin–ethylenediaminetetraacetic acid (EDTA) solution were obtained from Welgene, Daegu, Korea; bovine serum albumin (BSA) was obtained from GenDEPOT, Barker, TX, USA; and n-butanol was obtained from J.T. Baker, Mexico City, Mexico. Normal goat serum and primary and secondary antibodies were obtained from Abcam, Cambridge, MA, USA; Thermo-Fisher Scientific, Carlsbad, CA, USA; Santa Cruz Biotechnology, Santa Cruz, CA, USA; and AMRESCO, Solon, OH, USA.