Iron dextran

Adult zebrafish (Danio rerio) AB and embryos were maintained according to standard procedures at 28 °C [23 ]. A 10-h light/14-h dark day light cycle was used. The tp53^zdf1/+ (AB) fish (Zebrafish International Resource Center, ZIRC) were in-crossed according to procedures reported by Berghmans et al. [14 (link)]. The genotype of the progeny was determined by restriction fragment length polymorphism (RFLP) (ZIRC) of the tail clip genomic DNA. The zdf1 allele harbors a single T-to-A point mutation in exon 7 that changes Met to Lys at residue 214 which gives the genotype tp53^M214K. The zebrafish tp53^M214K mutation is an ortholog to the human tp53^M246K (c.737T > A; p.M246K; exon 7). Only tp53^M214K/M214K were used in this study. The study was approved by the Institutional Animal Care and Use Committee (no. 10476). E. coli DH5a (Yeastern Biotech, Taiwan) and Vibrio vulnificus YJ016 [13 (link)] were grown in shaking Luria-Bertani (LB) broth at 37 °C. The S. iniae wild-type strain 9117 [24 (link)] was cultured at 37 °C in Todd-Hewitt medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.2% yeast extract (BD Biosciences, San Jose, CA, USA) and 2% proteose peptone (Sigma-Aldrich). Azacitidine (Vidaza^®) was from Celgene (Summit, NJ, USA). Cytochalasin D, iron dextran and deferoxamine (DFO) were from Sigma-Aldrich.

Iron-loaded mouse model was generated as described previously [24 (link)]. Seven-week-old male B6D2F1 mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). This part of the study was approved by the Department of Health, Hong Kong SAR, and the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong. The investigations conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the ethical policy of our institution is listed as being compliant with that of NIH (assurance number A5773-01). Mice were acclimatized for at least one week before experiments. The 8-week-old male B6D2F1 mice were given intra-peritoneal injection of iron dextran (50 mg iron/ml; Sigma, St Louis, MO, USA), at 3.1 mg per 25 g body wt per day on a 5 days/week schedule, for 13 weeks. Control mice received 10% dextrose (Sigma, St Louis, MO, USA). Mice were sacrificed by an overdose of isoflurane anaesthesia until lack of respiration for 5 minutes to ensure euthanasia, followed by cervical dislocation. Ventricular tissues were stored at -80°C until analysis.

The immunosuppression model of murine OPC was used, as described previously [18 (link)], with the following modifications: 1) Mice were divided into two groups: control (without treatment; n = 8) and iron-overload (treated with 10 mg/kg of body weight of iron-dextran (Sigma) intraperitoneally [19 (link)] at day −1; n = 8); 2) Tranquilizer chlorpromazine hydrochloride (10 mg kg⁻¹) was given to each mouse on day 0, before infection [20 (link)]. On day 5, the mice were euthanized; and tongue, stomach (upper half of the body including 5–7 mm of the esophagus), and small intestine (10 cm in length, recovered from the cecum side) were excised. The right lateral half of each mouse's tongue and whole samples of the stomach and small intestine were weighed and homogenized in PBS. Serial dilutions of tissue mixture of all mice were plated individually on YPD-agar plates (containing 50 μg ml⁻¹ uridine and streptomycin/penicillin) and incubated at 30^ᵒC for 48 h. The C. albicans burden in tissues was assessed by Cfu (colony forming unit) quantification.

Fish were previously anesthetized with Trichaine methanesulfonate and weighed. A single dose respectively of 350 μg/g body weight iron dextran (Sigma, 100 mg/mL), 30 μg/g body weight deferoxamine (DFO, Novartis), and 50 μ/g body weight 4-OH-Tempol (Sigma) was injected intraperitonealy. Injections were performed under a stereomicroscope (Leica) using Amilton 10-μL syringes, control animals were sham-injected with saline solution. At 4, 8, 12, 24, 48, and 72 hours after injection brains were removed from anesthetized animals and put in a previously weighed tube for iron measurement or immediately frozen in liquid nitrogen for the following RNA extraction.

Iron dextran, rabbit polyclonal antibody against 3-nitrotyrosine (anti-3-NT), 2,4-dinitrophenylhydrazine, rabbit polyclonal antibody against dinitrophenol, TIM, triethanolamine hydrochloride, NADH, α-glycerophosphate dehydrogenase, DL-glyceraldehyde 3-phosphate solution, N-ethyl-maleimide, N-acetyl-imidazole and butylated hydroxytoluene were purchased from Sigma (St. Louis, MO, USA). Monoclonal anti-3-NT adducts IgG was obtained from Millipore Corp. (Billerica, MA, USA). Chemiluminescence system was purchased from Pierce (Rockford, USA). Detection kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione peroxidase (GPx), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and nitric oxide were gained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). NAD(H) kits were purchased from Suzhou Comin Biotechnology Co., Ltd. (Suzhou, China). rabbit polyclonal antibody against TIM and protein A/G agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG was obtained from Thermo Fisher (Rockford, USA). All other reagents and chemicals were of analytical grade and purchased from a local reagent retailer.