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For the overexpression of zebrafish nrf2a, capped nrf2a RNA was synthesized from pCS2nrf2  using an SP6 mMESSAGE mMACHINE in vitro transcription kit (Ambion, Austin, TX).
In vitro synthesized mRNA of nrf2a, the functional ortholog of mammalian Nrf2 in zebrafish, was injected into 1-cell stage of zebrafish embryos, and the gene expression in the injected embryos at 8h after the injection was examined using 16KMZH chips, which contain 16,399 probes .
Overexpression of nrf2a in mRNA-injected embryos was confirmed by a real-time qPCR (Figure S1, 75.5-fold higher compared to uninjected embryos).
Furthermore, looking back on the lineup of genes that were upregulated by the overexpression of nrf2a (Table S3), two more related genes were found: taldo1 (transaldolase 1) and fbp1a (fructose-1,6-bisphosphatase 1a).
The result indicated that the expression levels of pgd andfbp1a (1.40- and 2.76-fold, resp.), but not pckl, pcxb, and taldol (1.21-, 0.75-, and 0.70-fold, resp.), were significantly upregulated by the overexpression of nrf2a (Figure 3(b)).
Weak DEM-induced expression of fbp1a was also observed in the gills, but this induction was independent of nrf2a genotypes.
The genes that were selected included gclm (glutamate-cysteine ligase, modifier subunit), gsto2 (glutathione S-transferase omega 2), gsr (glutathione reductase), sqstml (sequestosome 1), and keapla (kelch-like ECH-associated protein 1a), together with a well-studied nrf2a target, gstp1 (glutathione S-transferase pi 1).
In our microarray analysis, we also found 55 genes that were downregulated by the overexpression of nrf2a (Table S4).
In the present microarray analysis, the cut-off value for significant upregulation by the overexpression of nrf2a was set at a 1.5-fold change instead of a 2-fold change in the previous studies [19, 20].
(a) The gene expression of the indicated proteasome subunits in 8 h postfertilization (hpf) wild-type embryos injected with or without 100 pg of nrf2a mRNA at the 1- cell stage was analyzed by a real-time qPCR.
(b) The gene expression of the indicated enzymes related to glucose metabolism in 8 hpf wild-type embryos injected with or without 100 pg of nrf2a mRNA at the 1-cell stage was analyzed by a real-time qPCR.
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