Tiron

The 3 isogenic HCT116 human colon cancer cells (p53^+/+, p53^−/−, and p21^−/−) cells were cultured at 10⁵ cells/mL in 96-well plates. After 24 hours, cells were treated in triplicates with 20 µM caspase-3, caspase-8, caspase-9 inhibitors, or the universal caspase inhibitor for 1.5 hours followed with 20, 40, or 60 µg/mL GT treatment. Proliferation effects were studied 48 hours posttreatment using the Cell Titer 96 (Promega Corporation, Madison, WI) nonradioactive cell proliferation kit.
The proliferation assay is a dye compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT)-based method, which measures the ability of metabolically active cells to convert tetrazolium salt into a blue formazan product and the absorbance is recorded at 570 nm. In experiments that involved Tiron (Sigma-Aldrich, St Louis, Missouri), the cells were treated in triplicates with 1 or 2 mM Tiron for 1.5 hours followed by GT treatment.

Primary neonatal rat cardiomyocytes (NRCMs) were obtained from 1- to 2-day-old Sprague-Dawley rats [18 ]. Removed and digested the heart in PBS containing collagenase type II (Worthington, NJ, USA) and pancreatin (Sigma, MO, USA). Deserted the atria and great vessels. Minced and digested the ventricles with collagenase type II and pancreatin. Collected and cultured Cells from digestion in DMEM (GIBCO, Invitrogen Inc.) for 2–4 h to decrease fibroblasts and increase cardiomyocytes. Cultured the cardiomyocytes at 37 °C with 5% CO2. The cells were received treatment when the cells were grown to 80–90% confluence. Firstly, divided and treated the primary cardiomyocytes with PBS, Ang II (10⁻⁶ M; Sigma), tumstatin (69–88) (10⁻⁶ M) and Ang II + tumstatin (69–88), respectively. Secondly, the primary cardiomyocytes were divided and treated with PBS, Ang II, Ang II + tumstatin (69–88), diethyldithiocarbamate (DETC; 10⁻⁶ M; Sigma) and DETC + Ang II + tumstatin (69–88), respectively. Thirdly, divided and treated the primary cardiomyocytes with PBS, Ang II, Tiron (antioxidant; 10⁻⁵ M; Sigma) and Ang II + Tiron. All the reagents added into the medium ate the same time for 24 h.

TP (molecular weight = 360.4 and purity ≥98% by HPLC, as described in the product sheet supplied by Sigma-Aldrich (St Louis, Missouri), catalog No. T3652), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), proteinase inhibitor solution, dimethyl sulfoxide (DMSO), Tiron, and Triton X-100 were purchased from Sigma-Aldrich. Ox-LDL was purchased from the Beijing Solarbio Life Science Company (Beijing, China). RPMI-1640 medium, carboxyfluorescein diacetate succinimidyl ester, fetal bovine serum, and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, California). Cytokine and chemokine ELISA kits were obtained from R&D Systems (Minneapolis, Minnesota). An 8-isoprostane assay kit was purchased from Cayman Chemical (Ann Arbor, Michigan). Malondialdehyde (MDA) and superoxide dismutase (SOD) activity assay kits were obtained from Beyotime Biotechnology Institute (Shanghai, China). A protein assay kit and RIPA lysis buffer were supplied by Bio-Rad Laboratories (Hercules, California) and Thermo Scientific (Waltham, Massachusetts), respectively.