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Fig. 3
Aldh2 Is Responsible for 5-Nitrofuran Activity in Zebrafish (A) A predicted model of daidzin binding to zebrafish ALDH2b, based on key residues involved in the human ALDH2-daidzin (PDB 2vle) protein-ligand interaction (Lowe et al., 2008). The equivalent residues in zebrafish Aldh2b are shown. Human ALDH2′Zebrafish Aldh2 2b (Phe-459′Phe-457; Phe-170′Phe-186; Trp-177′Trp-193; Val-120′Val-136; Phe-296′Phe-312; Phe-292′Ile-308; Asp-457′Asn-473; Cys-303′Cys-319). (B) Aldh2 and PKC inhibitors prevent 5-nitrofuran activity in zebrafish. Examples of zebrafish embryos treated at 2 dpf with 20 μM of the ALDH inhibitors daidzin or DSF for 1 hr, or with 20 μM of the PKC inhibitors PKC412 or Ro318220, and then treated with 5 μM NFN1 or 0.1% DMSO alone for 2 days. Experiments were repeated at least three times, with n > 10 embryos per condition. (C) Examples of 2 dpf zebrafish embryos pretreated with DMSO, 30 μM of daidzin, or the PKC inhibitor GFX 109203X for 1 hr, and then treated with 50 μM nifurtimox for 7 hr. Punctate melanocytes are indicated. Experiments were repeated at least three times (n = 5-10 embryos per condition) and treatment-condition cohorts blind scored. (D) Daidzin alters background adaptation in zebrafish embryos. (Left) Images of fixed zebrafish embryos (5 dpf) treated with 0.1% DMSO or 10 μM daidzin, and shifted from a dark environment to a light environment (light), or vice versa (dark). The average percentage of melanin coverage (within the area indicated by the red dotted outline) for each treatment condition ± SD is indicated. (Right) Box plot of melanin coverage (y axis) for each embryo in different treatment conditions (x axis). Individual values taken from one of three experiments are shown as red circles. The box depicts the lower quartile and the upper quartile, with the median depicted by the intersecting line. Whiskers extend between the minimum and maximum of all the data. In DMSO-treated embryos, melanocytes are significantly contracted in the light and expanded in the dark (p < 0.001, n = 20 for each condition; ANOVA, 95% confidence interval [CI] 11.081[5.966, 16.195]). Zebrafish treated with daidzin contract their melanin in response to light environment but do not significantly expand their melanin in response to dark environments (95% CI 0.563[4.552, 5.677]). The experiment was repeated three separate times with embryos at 5 dpf (n = 5-20 embryos per condition) and once with embryos at 4 dpf (n = 10 embryos per condition). See also Figure S3.