Fig 2: Complementary action of serine uptake and synthesis in challenged chondrocytes. Relative serine (Ser; a) and glycine (Gly; b) uptake in cultured chondrocytes derived from wild-type (Phgdhchon+) and chondrocyte-specific PHGDH knockout (Phgdhchon−) mice (n = 4–5). c Slc1a4 and Slc6a9 mRNA levels in wild-type and PHGDH-deficient chondrocytes (n = 3). d Growth curve of chondrocytes cultured in complete growth medium or serine/glycine-free medium (n = 3), based on their DNA content. e Proliferation of chondrocytes cultured in complete growth medium or serine/glycine-free medium (n = 3), as evidenced by BrdU incorporation. f Glucose uptake in chondrocytes cultured in complete growth medium or serine/glycine-free medium (n = 4–5). Serine (g) and glycine (h) labeling from 13C6-glucose in chondrocytes cultured in complete growth medium or serine/glycine-free medium (n = 6). Specific mass distribution vectors for each metabolite are shown. i Glut1, Phgdh, Psat1, and Shmt1 mRNA levels in chondrocytes cultured in complete growth medium or serine/glycine-free medium (n = 3). j Intracellular levels of the indicated metabolites in chondrocytes cultured in complete growth medium or serine/glycine-free medium (n = 4-5). R-5-P represents ribose-5-phosphate, Cit represents citrate, αKG represents α-ketoglutarate, Suc represents succinate, Fum represents fumarate, Mal represents malate and Asp represents aspartate. The data are presented as the means ± SDs; *P < 0.05 vs. Phgdhchon+, **P < 0.01 vs. Phgdhchon+, ***P < 0.001 vs. Phgdhchon+ (Student’s t test)

Fig 3: The serine synthesis pathway in growth plate chondrocytes. a Phgdh, Psat1, Shmt1, Shmt2, Mthfd1, and Mthfd2 mRNA levels in cultured skeletal progenitor cells (SPCs) and growth plate chondrocytes (CHs) (n = 3). b Immunoblot of PHGDH, PSAT1 and β-actin in SPCs and CHs (n = 3). c Immunoblot of PHGDH, PSAT1 and β-actin in SPCs and CHs after transduction with a lentiviral vector carrying a SOX9 overexpression (OE) plasmid or a shRNA (KD) against SOX9 (n = 3). Empty vector or scrambled shRNA was used as the respective control. d Schematic of carbon atom (circles) transitions of 13C6-glucose used to detect label incorporation into the shown metabolites. e Fractional contribution (FC) of 13C6-glucose (13C6-Glc) to serine (Ser) and glycine (Gly) in SPCs and CHs (n = 3). Serine (f) and glycine (g) labeling from 13C6-glucose in SPCs and CHs (n = 3). Specific mass distribution vectors (MDVs) for each metabolite are shown. h Intracellular Ser and Gly levels in SPCs and CHs (n = 3). The data are presented as the means ± SDs; *P < 0.05 vs. SPCs, **P < 0.01 vs. SPCs, ***P < 0.001 vs. SPCs (Student’s t test)

Fig 4: Genetic loss and pharmacological inhibition of PHGDH impairs GC responses.(A) Mice were injected with sheep RBCs and spleens examined by IHC and flow cytometry 8 days after immunization. (B) Representative flow cytometric analysis of splenic B cells from Phgdh+/+;Cd19-Cre (WT) or Phgdhfl/fl;Cd19-Cre (F/F) before and after immunization to identify GC B cells (CD19+B220+CD38loCD95hi), as well as DZ (CD86loCXCR4hi) and LZ (CD86hiCXCR4lo) B cells, within GC splenic population. (C) Flow cytometric analysis of absolute numbers of B cell subsets within CD19+B220+CD38loCD95hi splenic population from Phgdhfl/fl;Cd19-Cre (n = 6) and Phgdh+/+;Cd19-Cre (n = 4) after immunization. (D) Average GC area (left) and proportion (%) of GC area per spleen area (right) from Phgdhfl/fl;Cd19-Cre (F/F) and Phgdh+/+;Cd19-Cre (WT) mice after immunization. (E) WT mice were immunized with sheep RBCs 1 day before PH-755 treatment (300 mg/kg PH-755 orally twice daily for 7 days). Spleens were analyzed 8 days after immunization. (F) Representative flow cytometric analysis of splenic B cells from mice immunized with sheep RBCs and treated with vehicle/PH-755 to identify GC, DZ, and LZ B cells within GC splenic population. (G) Flow cytometric analysis of absolute number of GC, DZ, and LZ B cells within B220+ splenocytes collected from mice 8 days after sheep RBCs immunization; mice were treated with vehicle (n = 5) or PH-755 (n = 5). (H) Average GCs area (left) and proportion (%) of GC area per spleen area (right) from vehicle- and PH-755–treated mice 8 days after sheep RBC immunization. (I) Summary of NP2-specific plasma cells (PCs; left) and NP2-specific IgG1 PCs (right; total number per popliteal lymph nodes). WT mice were treated with either vehicle (n = 5) or PH-755 (n = 5) for 7 days. Animals were injected with NP-CGG 1 day before PH-755 treatment. Popliteal lymph nodes were collected 8 days after NP-CGG immunization. (J) Serum antibody titers for NP2-specific IgG1 8 days after NP-CGG immunization. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Mann-Whitney U test (C, G, I, and J) or unpaired t test (D and H).

Fig 5: Genetic loss and pharmacological inhibition of PHGDH reduces lymphoma progression in vivo.(A) Immunoblot of PHGDH, PSAT1, and PSPH expression in splenic B cells from WT (n = 3) and Eµ-Myc mice (n = 6). HSC70 was used as loading control. (B) Representative IHC staining (×20 magnification) for B220, Ki67, MYC, PHGDH, and PSAT1 abundance in sections of spleens from either WT (n = 3) or Eµ-Myc (n = 3) mice. (C) Isotope tracing analysis in splenic B cells isolated from either C57BL/6J WT mice or Eµ-MYC mice and cultured for 2 hours with 13C6-labeled glucose. Serine and glycine levels were measured by LC-MS. The percent distribution of each isotopologue of their respective metabolite pool is represented as mean ± SEM of triplicate cultures and is representative of 3 independent experiments. (D) Schematic showing lymphoma transplantation model, in which Myc/+;Rosa26-CreERT2/+;Phgdhfl/fl lymphoma cells are injected via the tail vein into 9-week-old male C57BL/6J mice. Three days after lymphoma engraftment, mice were randomized to receive either vehicle or tamoxifen treatment by oral gavage for 4 days. Samples were collected 20 days after injection. (E) Representative pictures of spleens from mice (n = 3 per group) sacrificed 20 days after transplantation (left), and quantification of the spleen weight (right). (F) Schematic showing lymphoma transplantation model, in which Myc/+ lymphoma cells are injected via the tail vein into 9-week-old male C57BL/6J mice. Two days after lymphoma engraftment, mice were randomized to be treated with either vehicle or PH-755 by oral gavage for 14 days. (G) Representative pictures of spleens from mice (n = 3 per group) sacrificed after 7 or 14 days after transplantation (left), and quantification of the spleen weight (right). Data are shown as the mean ± SEM. *P < 0.05 and ***P < 0.001, by 2-tailed Student’s t test (E and G).