Sin 1

Rabbit anti-CLDN1, mouse anti-CLDN4, rabbit anti-ZO-1, Alexa Fluor 488 anti-mouse, and Alexa Fluor 555 anti-rabbit antibodies, and H₂DCFDA were obtained from Thermo Fisher Scientific (San Diego, CA, USA). Goat anti-β-actin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-phosphoserine (p-Ser) and anti-phosphothreonine (p-Thr) antibodies were from Sigma-Aldrich (Saint Louis, MO, USA). LY, NiSPY-3, Quest Fluo-8 AM, rabbit anti-nitrotyrosine, rabbit anti-NOS1, rabbit anti-NOS2, rabbit anti-NOS3, and rabbit anti-ubiquitine were from Biotium (Fremont, CA, USA), Goryo Kagaku (Hokkaido, Japan), AAT Bioquest (Sunnyvale, CA, USA), R&D Systems (Minneapolis, MN, USA), Cell Signaling Technology (Beverly, MA, USA), ProteinTech (Rosemont, IL, USA), GeneTex (Irvine, CA, USA), and Stressgen Biotechnologies Corporation (British Columbia, Canada), respectively. NOC12, SIN-1, and L-NAME were from Dojindo Laboratories (Kumamoto, Japan). AMG9810, olvanil, and RN1734 were from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). DAF-2DA and FeTPPs were from Cayman Chemical (Ann Arbor, MI, USA). All other reagents were of the highest grade of purity available.

DNA cleavage was analyzed by detecting the conversion of the SC form of the pUC19 plasmid DNA (Invitrogen, Waltham, MA, USA) to the OC and linear forms. DNA (50 ng) was incubated with 0.1 mM SIN-1 (DOJINDO, Kumamoto, Japan) [26 (link),27 (link)] in the presence or absence of betanin in 50 mM sodium phosphate buffer (pH 7.4) for 1 h at 37 °C in the dark. Because of the low water solubility of catechins, a 20% acetone solution was used for dissolving catechin. The amount of acetone added corresponded to a final concentration of 0.1% (v/v). To exclude the effect of acetone, acetone was also added to a final concentration of 0.1% to the catechin-free control and SIN-1 groups. After incubation, DNA was purified using the FastGene Gel/PCR extraction kit (Nippon Genetics Co. Ltd., Tokyo, Japan). The DNA was collected and analyzed using 1% agarose/TBE gels. After electrophoresis, the gels were stained with 0.5 μg mL⁻¹ of ethidium bromide for 30 min and then photographed under UV illumination. Signal intensity was calculated using the ImageJ software [28 (link)].