Fig 2: Blocking PHGDH methylation with a synthesized nonmethylated peptide inhibits HCC growth.a The aa sequence of the TAT-methylated and TAT-nonmethylated peptides. b, c Endogenous PHGDH was immunoprecipitated in Huh7 and PLC/PRF/5 cells treated with the methylated or nonmethylated peptides (20 μM) for 24 h. Immunoblotting was performed with indicated antibodies (b). PHGDH activity was measured and normalized to PHGDH protein (c). Data in c are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t test. d, e Total serine (d) and glycine (e) levels in cells grown in -SG medium with treatment of the methylated or nonmethylated peptides (20 μM) for 24 h. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. f Growth rates of Huh7 and PLC/PRF/5 cells grown in CM or -SG medium with treatment of the methylated or nonmethylated peptides (20 μM). Data are presented as the mean ± SD (n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. g–i Images (g), weight (h), and volume (i) of HCC PDX tumors from mice fed with a -SG diet and treated with the methylated or nonmethylated peptides. Data are presented as the mean ± SD (n = 6 mice). Statistical analysis in h was performed using the two-tailed Student’s t test, and statistical analysis in i was performed using the two-way ANOVA with Bonferroni correction. j Representative images of IHC staining for Ki-67 in PDX tumors in g–i. Scale bars, 50 μm. k–m Images (k), weight (l), and volume (m) of Huh7 and PLC/PRF/5 tumor xenografts from mice fed with a -SG diet and treated with the methylated or nonmethylated peptides. Data are presented as the mean ± SD (n = 5 mice). Statistical analysis in l was performed using the two-tailed Student’s t-test, and statistical analysis in m was performed using the two-way ANOVA with Bonferroni correction. n Representative images of IHC staining for Ki-67 in Huh7 and PLC/PRF/5 tumor xenografts in k-m. Scale bars, 50 μm. Source data are provided as a Source Data file.

Fig 3: Arginine methylation at R236 elevates PHGDH catalytic activity by increasing substrate affinity.a Endogenous PHGDH was immunoprecipitated in cells treated with AMI-1 or AdOx for 24 h. The mono-methylation (me1) level of PHGDH was determined by immunoblotting. PHGDH activity was measured and normalized to PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. b MS identification of R236 mono-methylation of FLAG-PHGDH immunopurified by FLAG beads. c Sequence conservation of PHGDH protein using the Consurf coloring scheme. Maroon and cyan indicate high and low conservation, respectively. d HEK293T cells expressing GFP-PHGDH WT, R236K or V83A were treated with AdOx for 24 h. The mono-methylation (me1) level of immunoprecipitated GFP-PHGDH was determined by immunoblotting. e FLAG-PHGDH WT, R236K or V83A was expressed in HEK293T cells. The mono-methylation of immunopurified FLAG-PHGDH was measured using a site-specific antibody against R236 mono-methylation (mePHGDH (R236me1)), which was pre-incubated with the R236 mono-methylated (R236me1) peptide or the unmodified peptide before use. f Endogenous PHGDH was immunoprecipitated in cells treated with AMI-1 or AdOx for 24 h. The R236 mono-methylation level of PHGDH was determined by immunoblotting using mePHGDH (R236me1) antibody. g HEK293T cells stably expressing FLAG-PHGDH WT, R236K or V83A were treated with AMI-1 or AdOx for 24 h. The R236 mono-methylation level of immunopurified FLAG-PHGDH was determined by immunoblotting using mePHGDH (R236me1) antibody. The activity of immunopurified FLAG-PHGDH was measured and normalized to FLAG-PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. h, i FLAG-PHGDH WT, R236K or V83A was expressed in HEK293T cells and immunopurified by FLAG beads. The Km value of FLAG-PHGDH for 3-PG (h) or NAD+ (i) was determined. Data are presented as the mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig 4: PHGDH is hypermethylated at R236 by PRMT1 in human HCC Tissues.a, b Representative images (a) and quantitative analysis (b) of IHC staining using mePHGDH (R236me1) antibody in HCC tissues and paired normal tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t test. c, d Representative images (c) and quantitative analysis (d) of IHC staining for PRMT1 in HCC tissues and paired normal tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t-test. e, f IP and immunoblotting analysis with indicated antibodies in 20 HCC tissues (T) and paired normal tissues (N). WCL, whole cell lysates. R236 mono-methylation of immunoprecipitated PHGDH was determined and normalized to PHGDH protein (n = 20, cohort 2). g Representative images of IHC staining with mePHGDH (R236me1) and PRMT1 antibodies in HCC tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. h Pearson correlation test analyzing the relationship between the IHC staining intensity of mePHGDH (R236me1) and PRMT1 in HCC tissues (n = 42 samples, cohort 3). i Pearson correlation test analyzing the relationship between PHGDH activity and PRMT1 staining intensity in HCC tissues (n = 20 samples, cohort 2). j Pearson correlation test analyzing the relationship between PHGDH activity and the staining intensity of mePHGDH (R236me1) in HCC tissues (n = 20 samples, cohort 2). k, l Overall survival of HCC patients based on the IHC staining intensity of mePHGDH (R236me1) (k) or PRMT1 (l) (n = 30 samples). Statistical analysis was performed using the two-sided log-rank test. Source data are provided as a Source Data file.

Fig 5: R236 methylation of PHGDH mediated by PRMT1 promotes the growth of HCC cells.a–c PHGDH KO Huh7 cells were re-expressed with PHGDH WT, R236K (RK), or V83A (VA). Growth rates (a), colony formation assay (b), and quantification of clone numbers (c) of these cells grown in CM or -SG medium. Data in a are presented as the mean ± SD (n = 5 independent experiments), and statistical analysis was performed using the two-way ANOVA with Bonferroni correction. Data in c are presented as the mean ± SD (n = 3 independent experiments), and statistical analysis was performed using the two-tailed Student’s t-test. d–f Cells in a–c were subcutaneously inoculated into nude mice fed with a +SG or -SG diet. Tumor images (d), weight (e), and volume (f) were shown. Data are presented as the mean ± SD (n = 6 mice). Statistical analysis in d was performed using the two-tailed Student’s t-test, and statistical analysis in f was performed using the two-way ANOVA with Bonferroni correction. g, h Representative images (g) and quantitative analysis (h) of IHC staining for Ki-67 in tumor xenografts in d–f. Scale bars, 50 µm. Data in h are presented as the mean ± SD (n = 6 mice). Statistical analysis was performed using the two-tailed Student’s t test. i PHGDH KO plus PRMT1 KD Huh7 cells were rescued with PHGDH WT, R236K (RK) or V83A (VA), combining with or without PRMT1 re-expression. Growth rates of these cells grown in CM or -SG medium. Data are presented as the mean ± SD (n = 5 independent experiments), and statistical analysis was performed using the two-way ANOVA with Bonferroni correction. j–l Cells in i were subcutaneously inoculated into nude mice fed with a +SG or -SG diet. Tumor images (j), weight (k), and volume (l) were presented. Data are presented as the mean ± SD (n = 5 mice). Statistical analysis in k was performed using the two-tailed Student’s t-test, and statistical analysis in l was performed using the two-way ANOVA with Bonferroni correction. m, n Representative images (m) and quantitative analysis (n) of IHC staining for Ki-67 in tumor xenografts in j–l. Scale bars, 50 µm. Data in n are presented as the mean ± SD (n = 5 mice). Statistical analysis was performed using the two-tailed Student’s t test. Source data are provided as a Source Data file.