Figure 6. O-GlcNAcylation promotes DEN/CCl₄-induced hepatocellular carcinogenesis in PCK1-knockout mice.

(A) Reproductive strategy for generating AlbCre^(–/–), Pck1^(fl/fl) (WT), and AlbCre^(+/–), Pck1^(fl/fl) (liver-specific knockout, LKO) mice. (B) PCK1 protein expression in WT and LKO mouse organs involving the heart, liver, spleen, and kidney were confirmed by immunoblotting. (C) Schematic representation of the experimental procedures used with WT and LKO mice. Mice were injected intraperitoneally with 75 mg/kg DEN or 4% CCl₄ (every 3 days) as indicated, followed by combined administration of 5 mg/kg AOA or 1 mg/kg DON (twice per week) for 16 weeks. Control mice were provided a normal diet (ND). Gross appearances (D) and hematoxylin and eosin staining (E) (scale bar: 100 μm) of liver samples with tumors, and the numbers of tumor nodules (F), n = 6/group. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Tukey’s test. The yellow dotted-line circles represent tumors. AST (G) and ALT (H) levels in mouse serum samples (n = 6/group). (I) Glycolysis-metabolite profiles, derived from liver tumors of WT or LKO mice, were determined by performing LC-MS/MS metabolomics assays. (J) Relative UDP-GlcNAc levels (n = 6/group). The indicated proteins in liver tumors were assessed by immunohistochemical labeling (K) (scale bar: 100 μm) and Western blotting (L).