Fig. 19

The N2 neutrophils and liver injury phenotype also were observed in female ATP7B-KO mice. (A) Flow cytometric analysis of N1 (CD11b+Ly6G+NOS2+) and N2 (CD11b+Ly6G+CD206+) neutrophil populations in total CD11b+Ly6G+ cells derived from the livers of female wild-type and ATP7B-KO mice. (B) Flow cytometric quantification of CD11b+Ly6G+ neutrophils from female wild-type and ATP7B-KO mice livers. (C) Flow cytometric quantification of liver N1 and N2 neutrophils from female wild-type and ATP7B-KO mice. (B and C) n = 6 mice/group; unpaired 2-tailed t test. ∗∗∗P < .001, ∗∗∗∗P < .0001. (D) Pathologic changes in liver sections from female wild-type and ATP7B-KO mice at age 16 weeks. Upper panel: Representative H&E images in paraffin-embedded liver sections. Scale bar: 50 μm. Lower panel: Representative images of Sirius red staining in paraffin-embedded liver sections. Scale bar: 100 μm. (E) qPCR of mRNA levels of Tgfβ1 in liver hepatocytes isolated from female wild-type and ATP7B-KO mice. n = 3 sets of liver hepatocytes pooled from 2∼3 mice. ∗∗P < .01. (F) qPCR of mRNA levels of Stat3 in liver neutrophils isolated from female wild-type and ATP7B-KO mice. n = 3 sets of liver neutrophils pooled from 2∼3 mice. ∗∗P < .01. (G) Methylation-specific PCR in Socs3 promoter in liver neutrophils from female wild-type and ATP7B-KO mice. Left panel: Representative images of methylation-specific PCR in Socs3 promoter. Right panel: Quantification of band levels by ImageJ. M, methylated; U, unmethylated. n = 3 sets of liver neutrophils pooled from 2∼3 mice. ∗∗∗P < .001. WT, wild-type.