Fig 2: NNMT regulates intracellular methylation potential and CD44 transcription by inhibiting histone H3K27 methylation. (A) Mass spectrometric detection of relative SAM and SAH content and their ratio in NNMT-KD SMMC-7721 and NNMT-MHCC-97H cells. (B) Mass spectrometric detection of relative SAM and SAH content and their ratio in EV (empty vector), shNNMT, shNNMT + GNMT, GNMT and NNMT + GNMT cells. (C) GNMT protein expression in HCC cell lines, liver cancer tissues, and paracancerous tissues. (D) The effect of GNMT/NNMT overexpression on the expression levels of the other in 7721 cells. (E) Regression analysis of GNMT and NNMT mRNA expression in hepatoma cell lines. (F–G) H3K27me3 levels in the NNMT-overexpressing and KD cells. (H) CD44 mRNA levels in control and NNMT-KD SMMC-7721 cells after treatment with GSK-126 (5 mm for 72 h). (I–J) ChIP-qPCR analyses of H3K27me3 binding on CD44 promoter in NNMT-MHCC-97H and NNMT-KD SMMC-7721 cells. The t-test was used in A, B, D, H, I, G. The error bars represent SD. Each experiment was performed in triplicate. ***P < 0.001, **P < 0.01, and *P < 0.05

Fig 3: Critical role of GNMT downregulation in the reprogramming of the methionine cycle in Ndrg3 LKO livers. (A) qRT-PCR analysis of the expression of genes of the methionine cycle and branch pathways in the livers of wild-type and Ndrg3 LKO mice at 8-10 weeks of age. Mice were fasted for 15 h or refed for additional 6 h before being subject to the gene expression analysis (n = 4–5 per group). (B) GNMT protein expression in the livers of wild-type and Ndrg3 LKO mice. Mice at the age of 2 months (n = 5 per group) and 4 months (n = 4 per group) were fasted for 15 h or refed for additional 6 h before being subject to the protein expression analysis. (C) Western blot analysis of GNMT expression in primary hepatocytes isolated from wild-type and Ndrg3 LKO mice at 2 or 3 months of age. (D) Illustrative scheme describing plausible molecular mechanisms for the induction of methionine cycle reprogramming and GSD phenotypes in Ndrg3 LKO livers. Activation of CBS and inhibition of BHMT by SAM are indicated by red lines. Gene expression was normalized using Gapdh as the reference. Data are presented as mean ± SEM. p-values were calculated using two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. wild mice.