Fig 2: SHMT1 mediates the loss of nucleotide labeling induced by PHGDH inhibitionAll data are the mean of three biological replicates. Error bars represent standard deviations. *, p<0.05, Student’s t-test. a, NCT-503 induces increased synthesis of M+2 serine from M+2 glycine and unlabeled 5,10-CH2-THF in a PHGDH-dependent cell line. b, Probable SHMT1-catalyzed synthesis of M+1-serine from unlabeled glycine and 13C-serine-derived 5,10-methylene THF (5,10-CH2-THF) increases with PHGDH inhibition (10 µM NCT-503) and is suppressed by exogenous unlabeled formate. c, Serine synthesis pathway activity, or a serine synthesis pathway intermediate, represses SHMT1 activity. SHMT1 catalyzes serine synthesis from glycine and 5,10-CH2-THF. d, SHMT1 deletion restores incorporation of carbon from U-13C serine into dTMP in the presence of PHGDH inhibitor. Mouse SHMT1 expression restores decreased dTMP labeling induced by PHGDH inhibition. SHMT2 knockout does not block PHGDH inhibitor-mediated loss of dTMP labeling. e, SHMT1 deletion restores incorporation of carbon from U-13C serine into AMP in the presence of a PHGDH inhibitor. Mouse SHMT1 restores PHGDH inhibitor-mediated loss of AMP labeling by U-13C serine. f, NCT-503 treatment induces G1/S cell cycle arrest in MDA-MB-468 cells, consistent with a defect in nucleotide synthesis. g, Nucleoside supplementation partially rescues PHGDH inhibitor toxicity. h, Model of one-carbon unit wasting induced by PHGDH inhibition. Suppression of PHGDH activity increases the activity of SHMT1, which consumes one-carbon units to resynthesize serine but reduces the availability of one-carbon units needed for purine and dTMP synthesis.

Fig 3: ILF3 promotes tumor growth in vivo. a Tumor growth curves of DLD1 (1 × 106) or HCT-116 (1 × 106) colon cancer cells with or without ILF3 overexpression. Cells were subcutaneously injected into nude mice (n = 6). The tumors were isolated at the end of the experiments. b Serine pathway gene expression in the tumor tissues of (a). qRT-PCR analysis was performed to measure the mRNA levels of serine biosynthesis pathway genes. c Measurement of the subcutaneous tumor growth of ILF3-KD DLD1 cells (1 × 107). n = 9/group. The data are presented as the means ± SD. d Immunoblot analysis of protein levels of ILF3, p-Erk, Erk, PHGDH and PSAT1 in the subcutaneous tumor tissues generated in (c). e Representative IHC images of ILF3, PHGDH, Ki-67 and cleaved-Caspase-3 staining in the subcutaneous tumor tissues generated in (c). Scale bars represent 50 μm. f SGOC pathway gene expression after ILF3 KD in subcutaneous tumor tissues generated in (c). The data are presented as the means ± SD. g Measurement of SGOC pathway metabolites in subcutaneous tumor tissues obtained from (c). The data are presented as the means ± SD. *P < 0.05; **P < 0.01. h Relative ratios of reduced to oxidized glutathione (GSH/GSSG) in subcutaneous tumor tissues from (c) determined by LC–MS/MS. i Tumor growth curves of tumors derived from DLD-1 cells that were subcutaneously injected into nude mice (n = 6). Mice were treated with or without indicated amount of selumetinib. Tumor growth curves are shown. The data are presented as the means ± SD. j Representative IHC staining for ILF3, PHGDH, Ki-67 and cleaved-Caspase-3 in tumor tissues from (h). Scale bars represent 50 μm.

Fig 4: Serine biosynthetic activity correlates with platinum responsiveness in A2780 resistant cells.a Generation of cis SSP cell line established after serine/glycine deprivation of cis cells, n = 3 biological replicates for cis cells, n = 4 biological replicates for cis carbo cells (with three technical replicates each time), repeated measures two-way ANOVA, data are represented as mean ± SD. b Representative western blot of serine biosynthetic enzymes, n = 4 biological replicates. c RNA-seq results of serine biosynthetic enzymes and main transporters in cis SSP cells, mean values are plotted, n = 3 technical replicates, p values obtained by DESeq2 (Wald-test with Benjamini and Hochberg multiple testing). d Representative western blot of PHGDH and ATF4 in cis cells, serine and glycine starved cis cells and SSP cells, n = 2 biological replicates. e GI50 curves of wt, cis and cis SSP cells, n = 3 biological replicates (with three technical replicates each time), unpaired two-tailed t-tests between GI50 values, data are represented as mean ± SD, p = 0.0047 for cis vs. cis SSP +S/G and p = 0.0047 for wt vs. cis SSP -S/G. f Western blot of PHGDH and CD-PHGDH in genetic overexpressing cis cells, n = 3 biological replicates for cis-PHGDH and n = 2 biological replicates for cis-CD-PHGDH. g GI50 cuves of cis-control and cis-PHGDH overexpressing cells and cis-CD-control and cis-CD-PHGDH overexpressing cells, n = 4 biological replicates for cis-empty and cis-PHGDH and n = 3 biological replicates for cis-CD-empty and cis-CD-PHGDH (with three technical replicates each time), unpaired two-tailed t-tests between GI50 values, data are represented as mean ± SEM, p = 0.022 for cis-PHGDH vs cis-ctrl. Source data are provided as a Source Data file. Cis SSP serine synthesis pathway active cis cells, S serine, G glycine, CD catalytic dead.

Fig 5: PHGDH knockdown alters liver lipid profiles. a Volcano plot of lipidomics analysis of shPHGDH (N = 11) serum compared to shREN (N = 12). Significant metabolites are in bold. Triacylglycerol species are indicated in red. b Volcano plot of lipidomics analysis of shPHGDH (N = 11) liver compared to shREN (N = 11). Significant metabolites are in bold. Triacylglycerol species are indicated in red. c, d Total triacylglycerol (TAG) levels in the serum (c) and liver (d) of shREN and shPHGDH mice. Levels are normalized to shREN. e Individual TAG species in the liver of shPHGDH mice compared to shREN. Levels are normalized to shREN. f Fatty acyl carnitine (Car) levels in the liver of shREN and shPHGDH mice from the analysis in (b). Only significant (p < 0.05) metabolites are shown