(A) Schematic diagram for the conversion of U-[¹³C]-glutamine into various metabolites and LC-MS profiles of M+4 OAA, M+2 3PG, M+1 methionine, M+1 SAM, respectively, after PCK1-OE cells were incubated with U-[¹³C]-glutamine for 24 hours (n = 4 biologically independent samples). (B) Schematic diagram for the conversion of U-[¹³C]-pyruvate into various metabolites and LC-MS profiles of M+2 3PG, M+2 serine, M+1 methionine, M+1 SAM, respectively, after PCK1-OE cells were incubated with U-[¹³C]-pyruvate for 24 hours (n = 4 biologically independent samples). (C) Schematic overview showing that PCK1-induced H3K9me3 modification depends on SAM accumulation derived from SSP. (D) Intracellular SAM in PKO cells in the presence or absence of 3PG (n = 6 biologically independent samples). (E) PKO cells were treated with PEP (0.5 mM), 3PG (0.75 mM), serine (400 μM), methionine (100 μM), SAM (50 μM), and SAH (50 μM), respectively, for 24 hours. Immunoblots for H3K9me3 were repeated 3 times independently with similar results. Data from 1 representative experiment are shown. Densitometric analysis of H3K9me3 was performed, normalized to histone H3. (F) Immunoblots for H3K9me3 modification in SK-Hep1 cells. Data from 1 representative experiment are shown (n = 3). (G) Histone methyltransferase (HMT) activities for H3K9 in PKO cells (left) and PCK1-OE cells (right) (n = 3 technical replicates). (H–J) PKO cells or PCK1/SUV39H1 double-KO cells (PKO/SUVKO cells) were treated with or without SAM for 24 hours, and (H) Western blot (n = 3), (I) cell growth curves (n = 3 technical replicates), and (J) colony formation assays, Transwell assays, and wound scratch assays (n = 3 biologically independent samples), are shown. Data are shown as the mean ± SEM. Statistical analysis was performed using 2-tailed unpaired Student’s t test (A and B), 1-way ANOVA with Tukey’s test (D–H and J), or 2-way ANOVA with Bonferroni’s test (I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.