Fig 2: 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

Fig 3: dSA or Sph accumulation increases PHGDH expression and serine biosynthesis via ROS production. A: Generation of intracellular serine from glycolytic intermediates after 24 h treatment with dSA. The left panel shows intracellular levels of endogenously derived serine (m + 0), the right panel shows serine (m + 3) synthesized from labeled glucose. B: Increased levels of PHGDH in Sph or dSA-treated cells. Cells were cultured for 24 h in serine-reduced media (11 mg/l) containing increasing levels of dSA. PHGDH levels were then characterized by Western blot. The 1st two lanes show the effect of SG starvation on PHGDH levels and are included as a positive control. Also shown is the densitometry of the PHGDH band normalized to its corresponding actin band and presented as a percentage of the control, serine-replete cultured cells. C: Effect of myriocin on PHGDH upregulation. Myriocin (100 nM) was added at the same time as the medium was changed. Cells were processed 24 h later, and PHGDH levels were examined by Western blot. D: Addition of either dSA or Sph on PHGDH expression in both MOLT-4 and HEK293T. E: Role of de novo synthesis of sphingolipids on induction of PHGDH in response to serine deprivation. Myriocin (100 nM) was added concurrently with either serine-replete or serine-deficient media and incubated for 24 h before PHGDH levels were analyzed by Western blot. F: Increased levels of glycolytically derived glycine after treatment with Sph or dSA. The left panel shows intracellular levels of exogenously derived glycine, the right panel shows glycine synthesized from serine hydrolysis. Serine and glycine levels were measured by GC/MS after labeling cells with [13C6]-glucose for 24 h. G: Levels of PHGDH mRNA after treatment with NAC. Cells were medium changed to one containing reduced serine (labeled as < ser). At the same time, Sph, dSA, and NAC were added. After 24 h incubation, cells were prepared for qRT-PCR for PHGDH. Samples were analyzed in triplicate, and the mean average ± SD is shown. Statistical analysis for non-NAC treated cells was compared with the non-NAC treated control, while NAC-treated cells were compared with NAC-treated control. H: Effects of NAC on PHGDH protein levels. Cells were cultured for 24 h in serine-reduced media, containing either NAC or Sph or dSA. I: Effect of NAC treatment on the abundance of glucose-derived serine. NAC was added to cells for 3 h before the medium was changed to one containing [13C6]-glucose. The cells were incubated for a further 24 h before analysis of serine by HPLC GC/MS. HPLC GC/MS data are the mean average of five separate measurements, ± SD. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Fig 4: Metformin inhibits 2-HG production by PHGDH in MCF-7 cells without IDH mutations. (A) DNA sequencing against IDH1 R132 and IDH2 R172 residues was performed using genomic DNA from MCF-7 cells. Codons for IDH1 R132 and IDH2 R172 are indicated in gray boxes as CGT and AGG encoding Arginine. (B) The PHGDH level after metformin treatment was measured for protein (left) and mRNA (right) by Western blot and real-time PCR, respectively. (C) The alteration of the 2-HG level by PHGDH siRNA was measured with LC/MS. The data are from five independent experiments. The error bars represent the standard deviation.

Fig 5: Upregulation of the de novo serine synthesis pathway in Plasmax.A Serine can be synthesised de novo from 3-phosphoglycerate (3-PG) derived from glucose by a 3-step enzymatic reaction. The first is an NAD+-dependent reaction catalysed by PHGDH which converts 3-PG into 3-phosphohydroxypyruvate (3-PHP). 3-PHP is then converted by PSAT1 into 3-phosphoserine (3-PS) which is subsequently hydrolysed into serine by PSPH. B Percentage of pool of intracellular serine measured by LC–MS after incubation for 4 h with 22.5 mM or 5.5 mM of U-13C-glucose in DMEM or Plasmax, respectively. Data are represented as mean ± SD of triplicate wells. C Western blot of SSP enzymes of cells grown in DMEM (D) or Plasmax (P) for 72 h. Membrane was reprobed with Vinculin as a loading control. Blots are representative of 2 independent experiments. D Western blot of ATF-4 pathway of cells grown in DMEM (D) or Plasmax (P) for 72 h. Membrane was reprobed with Vinculin as a loading control. Blots are representative of 3 independent experiments. E Sum of ATP M + 6, ATP M + 7, ATP M + 8, ATP M + 9 percentage of pools measured by LC–MS after incubation for 4 h with 22.5 mM or 5.5 mM of U-13C-glucose in DMEM (grey) or Plasmax (blue). Data are represented as mean ± SD of triplicate wells (unpaired Student’s t test, ***p < 0.001). F Growth curve of cells grown in DMEM (grey) or Plasmax (blue) with or without 10 µM of PH755. Data are represented as mean ± SD of triplicate wells. G Percentage of M + 3 serine pools after incubation for 4 h with U-13C-glucose in DMEM (black), DMEM SG- (grey), Plasmax (light blue) or Plasmax SG- (dark blue) with or without serine/glycine (SG-). Data are represented as mean ± SD of triplicate wells (multiple comparison by one-way Anova, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). All graphs are a representative of 3 independent experiments. Source data are provided as a Source Data file.