Chaetocin

All animal experiments in this study were approved by the Keio University Institutional Animal Care and Use Committee (09088) and were carried out in accordance with the Institutional Guidelines on Animal Experimentation at Keio University. Five-week-old male DS rats were purchased from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). The rats were divided into four groups: control [normal-salt diet containing 0.3% NaCl, NS (−)], normal-salt diet with chaetocin [0.25 mg/kg of chaetocin (Sigma-Aldrich Co. LLC., St. Louis, MO, USA), NS (ch+)], HF [high-salt diet containing 8% NaCl, HS (−)], and treatment [high-salt diet with 0.25 mg/kg of chaetocin, HS (ch+)] group. chaetocin was dissolved in dimethyl sulfoxide (DMSO). Rats in the treatment group were administered 0.25 mg/kg of chaetocin intraperitoneally twice a week from 6 weeks of age until 13 weeks of age, as previously reported46 (link). Animals were sacrificed, and tissues were stored appropriately for individual experiments.

Human melanoma cell lines, Sk-Mel-28, A375, IGR37, LU-1205 and MV3 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and they were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a humidified incubator with 5% CO₂ at 37°C. Human primary melanocytes were obtained Kanglang Company (Shanghai, China) and cultivated in melanocytes growth with 10% FBS. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) and chaetocin were purchased from Sigma-Aldrich Corp. (St. Louis, MO). chaetocin was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution which was diluted to the final concentration with culture medium. The final concentration of DMSO was kept under 0.1% throughout the following studies, and showed no effect on cell morphology and proliferation in this study. The primary antibodies recognizing Bax, Bcl-2, procaspase-9/-3, cleaved caspase-9/-3, cytochrome c, PCNA, Nrf2, SOD2, catalase, and β-actin were purchased from Abcam Company (Cambridge, UK).

Six-week-old male BALB/c mice(n = 6 animals/group) were obtained from National Laboratory Animal Center(Taipei, Taiwan). The animals were housed in plastic cages and were provided Lab Diet 5001(PMI Nutrition International,. Brentwood, MO, USA) and water ad libitum during acclimatization. BALB/c mice were exposed to cigarette smoke using a cigarette smoke chamber equipped with whole-body exposure cages. The main-stream smoke from the combustion of 12 reference cigarettes(3R4F; Tobacco and Health Research Institute, KY, USA) were introduced into the whole-body exposure cages for a period of approximately 50 minutes/day, 5 days/week, for 12 weeks. Control mice were exposed to cigarette smoke-free HEPA-filtered air. The mass and number concentrations were monitoring using DusTrak(TSI DRX 8534) and P-Trak(TSI 8525) instruments, respectively. For the smoking/chaetocin group, smoking mice were given chaetocin(10 mg/kg body weight; Sigma-Aldrich Co. LLC.) by intraperitoneal injection on the final day. All animals were sacrificed at the end point after smoking challenge. The animal experiments were performed in compliance with the animal and ethics review committee of the Laboratory Animal Center at the Taipei Medical University, Taiwan(LAC-2014-0267).