Fig. 3

Figure 3. Generation of Nrf2-knockout zebrafish. (A) Gene knockout of zebrafish Nrf2 using CRISPR–Cas9 technology. CRISPR target sites were designed in exon 2 of nfe2l2a loci (blue arrowhead). In nfe2l2a^it321 line, 16-extra-amino-acids were added after the original Ile21 in nfe2l2a (diagonal stripes). (B) Expression of Nrf2-target genes in sulforaphane- or equol-treated larvae. Larvae at 3.5 dpf were treated without (Ctr) or with 40 µM sulforaphane (SF) or 25 µM equol (EQ) for 12 h in wild-type (WT), Nrf2-mutant (nfe2l2a^fh318), and Nrf2-knockout (nfe2l2a^it321) larvae, and the expression of gstp1.2 and prdx1 was analyzed using qRT-PCR. Letters A-F indicate significant differences (p < 0.05). (C) Antioxidant effects of equol and cinnamaldehyde in Nrf2-knockout larvae. nfe2l2a^it321 homozygous larvae at 3.5 dpf were pretreated without (gray, dotted) or with indicated phytochemicals (black, 25 µM equol or 50 µM cinnamaldehyde). After pretreatment for 12 h, the solution was replaced with 1.4 mM sodium arsenite (NaAsO₂), and survival was measured every 12 h for 48 h. p values of <0.01 were considered to indicate statistical significance.