2 7 dichlorodihydrofluorescein diacetate dcfh da

Ryanodine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]·cyclohepten-5,10-imine hydrogen maleate (MK-801), butylated hydroxyanisole (BHA), reduced glutathione (GSH), ascorbic acid (vit. C), cyclosporin A (CsA), dimethyl sulphoxide (DMSO), fetal calf serum and other materials for cell culturing, were purchased from Sigma Chemical Poland (Poznan, Poland). TBBPA (99.8 % purity) was synthesized and delivered commercially by the Institute of Industrial Organic Chemistry, Analytical Department, in Warsaw, Poland. Bastadin 12 synthesized in the Laboratory of Natural Products Synthesis and Bioorganic Chemistry, Institute of Bioorganic Chemistry NCRS “Demokritos” in Athens, Greece, was kindly provided by Dr. Emmanuel N. Pitsinos. Fluorescent probes fluo-3/AM, rhodamine123 (Rh123) and 2′7′-dichlorodihydrofluorescein-diacetate (DCFH-DA) were produced by Molecular Probes Inc. (Paisley, UK). All other chemicals were of analytical grade.

LAAO from B. leucurus venom was isolated and purified as described by Torres et al. (2010) [9 ]. DMEM medium, phenylhydrazone (FCCP), 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and catalase kit were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tetramethylrhodamine ethyl ester (TMRE), Fura-2AM and 2,7dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR, USA). AnnexinV/FITC Apoptosis Detection Kit was from BD Pharmigen (CA, USA). Z-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD), catalase and BAPTA-AM were from Tocris (Bristol, UK).

Rhodamine 123, propidium iodide, and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) utilized herein were acquired from Molecular Probes (Eugene, OR, USA). Rabbit polyclonal antibodies to cleaved caspase-3 (#9661), caspase-9 (#9502), γ-H2AX (#9718), and endonuclease G (#4969) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody for LC3B/MAP1LC3B (#NB600-1384) was from Novus Biologicals (Novus Biologicals, Littleton, CO, USA). Pan-caspase inhibitor (Z-VAD.fmk) and mouse monoclonal antibody specific for β-actin were from Sigma-Aldrich (Saint Louis, MO, USA). Monoclonal anti-poly (ADP-ribose) polymerase (PARP) antibody (#556494) and FITC annexin-V apoptosis detection kit were purchased from BD Pharmingen (San Diego, CA, USA).

MHY3200 was designed and synthesized by Prof. H. R. Moon at the College of Pharmacy, Pusan National University, Busan, Korea [7 (link)], and dissolved in dimethyl sulfoxide (DMSO). Other chemical reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes Inc. (Eugene, OR, USA). ECL Western Blotting Detection Reagents, Pierce bicinchoninic acid (BCA) protein assay kit, polyvinylidene difluoride (PVDF) membrane, and Dokdo-MARK™ protein size marker were purchased from GE Healthcare Biosciences (Buckinghamshire, UK), Thermo Fisher Scientific (Rockford, IL, USA), the Millipore Corporation (Bedford, MA, USA), and ElpisBiotech (Daejeon, Korea), respectively. Anti-NOX4, anti-p-Akt (Ser 473), manganese superoxide dismutase (MnSOD), p-p65 (Ser 276), p65, β-actin, transcription factor IIB (TFIIB), and cyclooxygenase-2 (COX-2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-FoxO1 (Ser 256) were purchased from Cell Signaling Technology (Danvers, MA, USA) while anti-rabbit IgG-horseradish peroxidase-conjugated antibodies, anti-mouse IgG-horseradish peroxidase-conjugated antibodies, and anti-goat IgG-horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Gyp was kindly provided by Ankang Pharmaceutical Institute of the Beijing University. The powder was dissolved in 80% ethanol (EtOH) to get a stock solution of 100 mg/ml, which was sterilized by a 0.22 µm membrane filtration. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT), rhodamine-123 (Rh123), Hoechst 33342, propidium iodide (PI) and N-acetylcysteine (NAC) were purchased from the Sigma Chemical Company (St. Louis, MO, USA). 2′, 7′- dichlorodihydrofluorescein-diacetate (DCFH-DA) and FITC-phalloidine were supplied by Molecular Probes Inc. (Carlsbad, CA, USA). Guava Nexin Reagent was obtained from Millipore Corporation (Billerica, MA, USA). All the other reagents were commercial products of analytical grade.

Lysophosphatidylcholine, baicalein, fura-2/AM, β-actin antibody and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Aldrich Chemical Company (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR). Antibodies to Bcl-2, Bax, ERK1/2, JNK and phosphorylated JNK were obtained from Upstate Biotechnology (Lake Placid, NY, USA) while antibodies of p38, phosphorylated p38 and cytochrome c were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Antibodies to cleaved caspase-3, cleaved caspase-9, and phosphorylated ERK1/2 were obtained from Cell Signaling Technology (Beverly MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture reagents were obtained from GIBCO BRL Life Technologies (Grand Island, NY, USA). In the experiments, baicalein was prepared by dissolving with DMSO. Dilutions were made in phosphate-buffered saline (PBS) and filtered through a 0.22 μM syringe filter.