Fig 1: Depletion of PHGDH-sensitized HCC cells to Sorafenib treatment. a Knockout of PHGDH in MHCC97L cells by CRISPR/Cas9 gene editing system. b Knockout of PHGDH showed mild effect on cell proliferation in in vitro cell culture. However, knockout of PHGDH significantly suppressed MHCC97L cell proliferation in the presence of Sorafenib (black connected dots: non-target control; red connected dots: sgPHGDH#2; deep blue connected dots: sgPHGDH#3; green connected dots: sgPHGDH#4; purple connected dots: sgPHGDH#12; light blue connected dots: sgPHGDH#32). c Knockout of PHGDH induced apoptosis significantly upon Sorafenib treatment (gray bar: vehicle-treated group; red bar: Sorafenib-treated group). d Knockdown of PHGDH by lentiviral-based shRNA approach. e Knockdown of PHGDH suppressed HCC cell proliferation under Sorafenib treatment (black connected dots: non-target control; red connected dots: shPHGDH#20; deep blue connected dots: shPHGDH#32). f Knockdown of PHGDH augmented Sorafenib induced apoptosis in HCC cells (gray bar: vehicle-treated group; red bar: Sorafenib-treated group). g Knockdown of PHGDH sensitized HCC cell to Sorafenib in nude mice (gray connected dots: Vehicle-NTC, non-target control treated with vehicle; blue connected dots: Sora-NTC, non-target control treated with Sorafenib; red connected dots: Vehicle-shPHGDH, PHGDH knockdown clones treated with vehicle; purple connected dots: Sora-shPHGDH, PHGDH knockdown clones treated with Sorafenib). The error bar in panels b, c, d, f represents the standard error of mean (SEM), n = 3 biologically independent samples. The error bar in panel g represents the standard deviation (SD), n = 6 mice. Source data are provided as a Source Data file. (Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001)
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
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