Fig. 5

(A) Schematic representation of exon 2 of the WT mouse Keap1 allele, the ssODN, and the W→A (replacement of C3IR of the mouse protein with that of zKeap1A) mutant allele after homologous recombination. The sgRNA and its protospacer adjacent motif are indicated by continuous and dotted overlines, respectively. The 5′ untranslated region and open reading frame of Keap1 are represented by the light and dark gray boxes, respectively. The mutation is shown in red. (B) GSEA plot performed for a gene set consisting of Nrf2 target genes with RNA-seq data obtained from the liver of 8-week-old Keap1^W→A/W→A and WT mice. NES, normalized enrichment score. (C) GO analysis of core enrichment genes in (B). The size of the bubbles indicates the number of genes enriched in the corresponding GO term, and their color represents the E ratio (48). (D) Experimental plan for assessment of liver injury induced by an overdose of APAP. (E and F) Representative hematoxylin and eosin staining of liver sections from APAP-treated Keap1^W→A/W→A or WT mice (E). The area of necrosis in such sections was quantified with ImageJ/Fiji (F). Quantitative data are means from eight or nine mice of each genotype. (G) Representative TUNEL staining for the liver of APAP-treated Keap1^W→A/W→A or WT mice. (H to J) Serum cholesterol ester/total cholesterol ratio (H) as well as AST (I) and ALT (J) activities for APAP-treated WT, Keap1^WT/W→A, or Keap1^W→A/W→A mice. Data are means for 8 or 10 mice of each genotype. (K) GSH/GSSG ratio for the liver of APAP-treated WT, Keap1^WT/W→A, or Keap1^W→A/W→A mice. Data are means for 8 or 10 mice of each genotype. (L) Immunohistochemical staining of 4-HNE in liver sections from APAP-treated Keap1^W→A/W→A or WT mice. Scale bars, 100 μm.