2 Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction 37 (Philomath, OR, USA). The sequences for the ctns and control morpholinos were ATTGTTCTGTCGTTCAGCTTAACGC and GGATTAAAATCCGCTACTCACATCC, respectively. 0.2 mM of either ctns or control morpholino with 0.1 mM p53 morpholino (GCGCCATTGCTTTGCAAGAATTG) to minimize apoptosis, were injected into the yolk sac of one cell stage embryos in a total volume of 1 nl, as previously described59. Success of injection was checked immediately under fluorescent microscopy. Evaluation of morphological changes was performed at 4 dpf, while homogenization for cystine assay was performed at 6 dpf. Cystine measurement Cystine content in homogenates of zebrafish larvae (6 dpf) or organs of adult zebrafish (8 months) of each genotype was evaluated using high performance liquid chromatography(HPLC)60. ctns‒/‒ larvae were either free of treatment (N=133) or subjected to 0.1 or 1.0 mM of cysteamine in the swimming water (N=111 and 121 larvae, respectively). The swimming water was refreshed with the specified concentrations of cysteamine starting from 3 hpf and every 24 hours. Cystine levels were compared with wt embryos (N=191) in two independent experiments. Groups of 20-40 embryos of each treatment condition and each genotype were homogenized together by sonication in 200 µl of 5mM N-ethylmaleimide (NEM) (Sigma, St Louis, MO, USA) in 0.1 M PBS. 100 µl of 12% sulfosalicylic acid (SSA) were added to each homogenate and samples were centrifuged at 12,000 g for 10min. Supernatants containing stabilized cystine were removed completely and preserved at −80°C until time of analysis, while pellets were dissolved overnight at 4°C in 300 µl of 0.1M NaOH then kept at −80°C until protein is measured. Adult organs of 8 months ctns‒/‒ or wt zebrafish (N=3 each), were dissected then homogenized in a similar way to larval samples. Glutathione measurement Oxidized glutathione (GSSG) was measured in homogenates of 6 dpf wt and ctns‒/‒ larvae with NEM similar to cystine, while total glutathione (GSH) and free cysteine were measured in larval homogenates without the addition of NEM (wt =158 larvae, untreated ctns‒/‒ =80 larvae, 0.1mM cysteamine treated ctns‒/‒ =108 larvae and 1.0mM cysteamine treated ctns‒/‒ =104 larvae). Immediately after preparation of 3-5homogenates of each condition, 12% SSA was added to prevent protein binding of glutathione. The homogenates were centrifuged at 12,000 g for 10 min at 4°C. The supernatants of corresponding samples were used for measurements of GSSG or GSH by HPLC60.All results were referred to protein measurements in pellets.
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