Figure 1.

GAN DIO-NASH-HCC mice show progressive development of severe NASH with advanced fibrosis. Mice were fed the GAN diet for 12–72 weeks (n = 15 per group). Chow-fed mice (n = 10) served as normal controls. (A) Study outline illustrating endpoints applied at all GAN diet-induction periods (plasma/liver biochemistry, NAS and fibrosis scoring, quantitative liver histology, liver (non-tumor) RNA sequencing, macroscopic tumor counts) and additional endpoints applied after 72 weeks of GAN diet feeding (tumor histology, tumor RNA sequencing, liver flow cytometry). (B) Terminal body weight. (C) Liver weight. (D) NAFLD Activity Score (NAS) and fibrosis stage. (E) Steatosis, lobular inflammation and ballooning degeneration scores. (F–J) GHOST-based histomorphometrics on histopathological scoring variables, including lipid-laden (F) hepatocytes, (G) inflammatory foci, (H) hepatocyte ballooning and (I, J) periportal/sinusoidal fibrosis. Lower panels: HE and PSR stainings illustrating the development of steatosis and perisinusoidal fibrosis in GAN DIO-NASH-HCC mice (scale bar, 100 µm). (K–O) Proportionate (%) area of (K) lipids, (L) inflammation (galectin-3), (M) α-SMA (fibrogenesis marker) and (N, O) fibrosis (PSR, Col1a1). See Fig. S1 for total liver histological marker levels. Lower panels: Representative photomicrographs of galectin-3, α-SMA, and Col1a1 immunostainings (scale bar, 100 µm). ***p < 0.001 versus chow-fed controls (Dunnett’s test one-factor linear model).