Characterization of sex disparity in xmrk and Myc-induced HCC progression. Three-month-old, xmrk+, Myc+ and wildtype zebrafish were treated with 60 μg/ml dox for 7 days. 10 fish were analysed in each group and the experiment was repeated multiple times. (A) Gross morphology of male and female fish (left lateral view). The livers are outlined. (B) Quantification of percentage of liver area to abdomen area. (C) Representative images of liver sections of male and female fish after H&E staining. (D) Quantification of tumor histology in male and female fish. *P < 0.05. Scale bars: 2 mm in (A) and 20 μm in (C).

Proliferation and apoptosis in the livers of male and female xmrk+ and Myc+ fish following oncogene activation. 10 fish were analysed in each group and the experiment was repeated multiple times. Proliferation and apoptosis were examined by PCNA and Caspase 3 staining respectively. (A) IF staining of PCNA in liver sections. (B) Quantification of densities of proliferating liver cells (PCNA+). (C) IF staining of Caspase-3 in liver sections. (D) Quantification of densities of apoptotic liver cells (Caspase 3+). *P < 0.05. Scale bars: 20 μm.

Determination of HSCs and activated HSCs in the livers of male and female xmrk+ and Myc+ fish following oncogene activation. 10 fish were analyzed in each group and the experiment was repeated once for reproducibility. (A) IF co-staining of GFAP (red) and a-SMA (green) in liver sections. White boxes indicate the enlarged area as shown on the right. (B) Quantification of total HSC density in liver sections. (C) Quantification of activated ratio of HSCs in liver sections. *P < 0.05. Scale bars: 20 μm.

Immunofluorescent staining for serotonin and P-Tph1 in the livers of male and female xmrk+ and Myc+ fish following oncogene activation. 10 fish were analyzed in each group and the experiment was repeated once for reproducibility. (A) IF co-staining of serotonin (red) and HNF4a (green) in liver sections. (B) Quantification of ratio of serotonin-productive hepatocytes in liver sections. (C) IF co-staining of P-Tph1 (red) and HNF4a (green) in liver sections. (D) Quantification of ratio of P-Tph1-expressed hepatocytes in liver sections. *P < 0.05. Scale bars: 20 μm. ns, non-significance.

Determination of neutrophils and macrophages in the livers of male and female xmrk+ and Myc+ fish following oncogene activation. 10 fish were analyzed in each group and the experiment was repeated once for reproducibility. (A) IF co-staining of Csf1r (red) and Lcp (green) in liver sections. (B) Quantification of neutrophil densities in liver sections. (C) Quantification of macrophage densities in liver sections. *P < 0.05. Scale bars: 20 μm.

Immunofluorescent staining for cortisol and Tgfb1a in the livers of male and female xmrk+ and Myc+ fish following oncogene activation. 10 fish were analyzed in each group and the experiment was repeated once for reproducibility. (A) IF co-staining of cortisol (red) and HNF4a (green) in liver sections. (B) Quantification of ratio of cortisol-expressing hepatocytes in liver sections. (C) IF co-staining of Tgfb1a (red) and HNF4a (green) in liver sections. (D) Quantification of ratio of Tgfb1a-expressing hepatocytes in liver sections.