Protease inhibitor mixture solution

Human colorectal carcinoma cell line, RKO, was obtained from American Type Culture Collection (Manassas, Virginia, USA). Minimum essential medium (MEM) was purchased from Gibco Laboratories (Paisley, UK). Fetal bovine serum (FBS) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma-Aldrich Chemical Company (Dorset, UK). Six-well flat-bottom tissue culture plates were purchased from Nunc Inc. (Hereford, UK). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) and protease inhibitor mixture solution was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Enhanced chemiluminescence (ECL) western blotting detection reagents and pure nitrocellulose MEMbranes were purchased from GE Healthcare (Buckinghamshire, UK). Primary antibodies for the detection of caspase 3 and β-actin and goat antirabbit and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich Co., Ltd. unless otherwise specified.

Protease inhibitor mixture solution and 10% neutral-buffered formalin were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR, USA). The pure nitrocellulose membrane was purchased from Bio-Rad Laboratories (Tokyo, Japan). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Rabbit polyclonal antibodies against insulin, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)-4, p22 phox , p47 phox and cyclin E, and mouse monoclonal antibodies against phosphor-c-Jun N-terminal kinase ( p-JNK), Bax, cytochrome c, β-actin, and histone were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit polyclonal antibody against pancreaticduodenal homeobox 1 (PDX-1) (LifeSpan BioSciences, Seattle, WA, USA) and mouse monoclonal antibody against caspase 3 (BioVision, Mountain View, CA, USA) were also used. Goat anti-rabbit and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. ECL western blotting detection reagents were purchased from GE Healthcare (Piscataway, NJ, USA). All other chemicals and reagents were purchased from Sigma Chemical Co.