Figure 1: Characterization of the urea cycle in HCC.
A, Metabolic gene set analysis of TCGA RNA-sequencing data in HCC tumors (n=374) and normal liver tissues (n=50), and 2752 genes encoding all known human metabolic enzymes and transporters according to the Kyoto Encyclopedia of Genes and Genomes (KEGG). Metabolic genes were ranked based on their median fold expression changes in HCC tumors vs normal tissue. B, Schematic overview of the hepatic urea cycle. C, RNA-seq reads of urea cycle genes in normal liver [L] and tumor tissues [T] from TCGA dataset. D, mRNA expression of urea cycle enzymes in healthy liver (n=9) and human HCC tumor samples (n=7). E, F, Representative micrographs of immunohistochemical staining (E) of urea cycle enzymes in patient HCC vs healthy liver samples and quantification (F). Scalebar = 100 μm. G, H, Western blot (G) and mRNA expression (H) analysis of urea cycle enzymes in HCC cell lines and human primary hepatocytes. I, Kaplan-Meier survival curve of average urea cycle TCGA gene expression (CPS1, ASS1, ASL, ARG1, and OTC) using KM-plotter with automatically selected cut-off of 50% (Nagy et al., 2018). J, Flow cytometric analysis of EdU incorporation in SNU-398 cells with ectopic ASS1 expression, as well as catalytically dead versions of ASS1 (Khare et al., 2021). K, EdU incorporation analyses in cultured SNU-398 (control and ASS1 expressing) cells treated with various doses of aspartate. Aspartate supplementation was combined with expression of the hEAAT1 surface protein. L, Growth curves of SNU-398 cells in control conditions and upon ASS1 re-expression in the presence of all four nucleosides. M, SNU-398 cell growth in control conditions and upon ASS1 re-expression in the presence of pyrimidines exclusively. GAPDH is used as loading control for all westerns in this study. For all figures, data are presented as mean ± SEM. **** or #### (p < 0.0001), *** or ### (p < 0.001), ** or ## (p < 0.01), * or # (p < 0.05). Student’s two-tailed unpaired t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, or log-rank test for comparisons of survival distributions of two groups. See also Figure S1.