PBS overnight at 4uC, and washed twice in PBS, once in 50% methanol, and twice in 100% methanol, and cooled to –20uC for at least 3 hours. Fixed larvae were then brought back to room temperature (RT), washed twice in PBT (0.1% Tween 20/PBS) for 5 minutes, and immersed for 2 hours in 9% hydrogen peroxide in PBT. After immersion, the larvae were washed twice in PBTw (0.2% bovine serum albumin in PBT), treated for 20 minutes with 50 mg/ml proteinase K (Sigma-Aldrich, St. Louis, MO), and fixed with 4% PFA/PBS for 20 minutes at RT. Microarray analysis A microarray analysis was performed using custom-made 16 K MZH chips. The MZH chips contained in a total of 16399 probes of a 65 oligonucleotides in length purchased from Sigma-Aldrich. The collected zebrafish embryos were quickly homogenized with 1 ml of QIAzol reagent (Qiagen, Hilden, Germany), and subsequently stored at -80uC. Total RNA was extracted according to the manufacturer’s instructions. Isolated total RNA was then further purified using the RNAeasy mini kit (Qiagen). Amino-allyl-modified amplified RNA was synthesized in one amplification round from 1 mg of purified total RNA using the amino-allyl RNA amplification kit (Sigma-Aldrich). Subsequently, 5 mg of amino-allyl-modified amplified RNA was used for coupling of monoreactive Cy3 and Cy5dyes (GE Healthcare, Little Chalfont, UK) and column purified. Dual-color hybridization of the MZH chips was performed according to the manufacturer’s instructions for the AceGene DNA microarray (Hitachi Solutions, Tokyo, Japan). Each experi- ment was repeated in triplicate. After hybridization, MZH chips were scanned using the Affymetrix 428 array scanner (Affymetrix, Santa Clara, CA). The microarray data were processed from raw data image files with Affymetrix Jaguar (Affymetrix) and normalized. The processed data were subsequently imported into Excel (Microsoft, Redmond, WA) to compare expression profiles of DEM-treated samples to control samples. The cut-offs for significance regarding the ratios of DEM-treated samples vs control samples were set at a 2-fold change. We have deposited the raw data at GEO under accession numbers (GSM799460, GSM799461, GSM799462, GSM799463), and we can confirm all details are MIAME compliant. Plasmid construction cDNA clones were prepared of the following transcripts by RT- PCR using total RNA from 5 days post-fertilization (dpf) zebrafish larvae: gstal, zgc:158387 (mgst3b), sepw2b, bcat1, zgc:110343 (prdx1), zgc:163022 (frrs1c), zgc:92066 (fthl), gclc, gclm, and hmox1 (hmox1a). Specific primers were designed based on cDNA information (http://zfin.org), and cDNA products were subcloned into the pBluescript II SK vector (Table S2). Plasmids pCS2nrf2, pCS2FLmKeap1, and pKSgstp1N have been described previously [8,9,12]. Knockdown and overexpression analyses of Nrf2 Synthetic capped nrf2 RNA was made with an SP6 mMES- SAGE mMACHINE in vitro transcription kit (Ambion, Austin, TX) using pCS2nrf2. The Nrf2-morpholino oligonucleotide (nrf2MO) has been described previously [9]. mRNA or morpho- lino oligonucleotides were injected into yolk of the zebrafish at the one-cell stage using an IM300 microinjector (Narishige, Tokyo, Japan). Sectioning of zebrafish larvae After carrying out WISH analysis, larvae were fixed with 4% PFA/PBS, embedded in 1.5% SeaPlaque GTG agarose (Takara Tissue-Restricted Induction of Nrf2 Target Genes Bio, Osaka, Japan), and dehydrated through graded ethanol series (30%, 50%, 90% and twice 100%) in PBS for 15 minutes each. Glycol methacrylate (Technovit 8100; Heraeus Kulzer, Wehr- heim, Germany), with low-temperature polymerization, was used according to the manufacturer’s instructions. After embedding, 6- mm serial sections were made from whole bodies of zebrafish larvae with an RM 2045 microtome (Leica, Wetzler, Germany). Results Identification of Nrf2 target genes in zebrafish In order to investigate tissue-specific expression of Nrf2 target genes, we searched for zebrafish Nrf2 targets other than gstp1 that were able to be used for WISH analysis. Microarray analysis was carried out using cDNA prepared from 4-dpf larvae treated with or without 100 mM DEM for 12 hours. In total, 16,000 zebrafish cDNAs were screened and 42 genes were identified that showed more than a 2-fold induction compared with DEM-treated larvae and untreated larvae (Table S3). The reliability of this screen was demonstrated by the fact that gstp1 produced a top ranking score in this analysis. In microarray analysis, gclm and nqo1 were induced by DEM at levels of only 1.97- and 1.93-fold, respectively. Since they have generally been used as Nrf2 target genes in mammalian cells, we selected them together with gclc and hmox1, for further analysis, in addition to 42 identified genes (Table S4). We next carried out RT-PCR analysis to confirm the results of microarray analysis. RT-PCR analysis was performed using RNA isolated from 4-dpf larvae treated with DEM and isolated RNA (Figure 1 and Table S3). Thirty-three genes were analyzed out of 46 selected genes, and 19 genes were identified to be induced by DEM. To clarify whether these inductions were directed by Nrf2, we carried out RT-PCR analysis using Nrf2-knockdown larvae Figure 1. Screening of Nrf2 target genes in zebrafish. DEM- induced expression of candidate genes for Nrf2 target analyzed by RT- PCR. Embryos were injected with or without nrf2MO and treated with or without 100 mM DEM for six hours at 2 hours post-fertilization (hpf) using total RNA from the whole bodies. doi:10.1371/journal.pone.0026884.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26884