Fig. 4

Effect of SIRT4 overexpression on methionine cycle and HCC tumorigenesis. A Heatmap displaying metabolite changes upon methionine restriction (MR). The red color indicates detected metabolites were increased while the blue ones decreased between MR and Ctrl conditions in six biological replicates. B Pathway enrichment analysis of differentially detected metabolites (fold change > 1.5, p-value < 0.05) upon methionine restriction in MetaboAnalyst 4.0. The circle color is indicative of the level of enrichment significance, with yellow being low and red being high. The circle size is proportional to the pathway impact value. C Schematic of the methionine cycle. D The changes of specific metabolites upon methionine restriction. Blue means increased while red indicates decreased. E Western blot analysis of modified histones in the cell culture supplemented with specific metabolites. Total histone H3 was used as a loading control. F, G Migration and sphere formation efficiency of primary liver cells derived from SIRT4^+/+ and SIRT4^−/− mice under control or methionine restriction conditions. Quantification was shown in the right panel. H Immunoblot showing SIRT4 protein level in the liver lysates of SIRT4^+/+ and SIRT4^−/− mice. I, J Tumour volumes and survival analysis of SIRT4^+/+ and SIRT4^−/− mice. n = 5 mice per group. Data are means ± SEM. Group differences were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test (F, G, I) or log-rank test (J) (**p < 0.01, ***p < 0.001 ****p < 0.0001). n.s., not significant