Fig. 7.

Combined deficiency of serine uptake and de novo serine synthesis severely affect chondrocyte function. a Intracellular levels of the indicated metabolites in chondrocytes isolated from wild-type (Phgdh^chon+) and chondrocyte-specific PHGDH knockout (Phgdh^chon−) mice and cultured in complete growth medium (+) or serine/glycine-free medium (−) (n = 3). Ser represents serine, Gly represents glycine, Cit represents citrate, αKG represents α-ketoglutarate, Suc represents succinate, Fum represents fumarate, Mal represents malate, R-5-P represents ribose-5-phosphate, Asp represents aspartate, Glu represents glutamate, Pro represents proline and GSH represents reduced glutathione. Proliferation (b; quantified by BrdU incorporation), ROS content (c; quantified by the CM-H₂DCFDA mean fluorescence intensity, MFI), viability (d; quantified by AnxV-PI flow cytometry, viable cells are AnxV^-PI^-) and matrix deposition (e; Alcian Blue (AB) staining) of wild-type and PHGDH-deficient chondrocytes cultured in complete (+) or serine/glycine (S/G)-free medium (−) (n = 3). f Schematic overview of the experimental setup. Chondrogenically primed control (PHGDH^ctrl) or PHGDH-deficient (PHGDH^KD) progenitor cells were ectopically implanted into mice fed an amino acid (AA) control diet or a serine/glycine-free diet. Ossicles were collected 7 days after implantation. g Immunoblot of PHGDH and β-actin in PHGDH^ctrl and PHGDH^KD cells prior to implantation (n = 4). Volume (h) and cell density (i) of PHGDH^ctrl and PHGDH^KD ectopic implants (n = 4). j COL2 immunostaining in PHGDH^ctrl and PHGDH^KD ectopic implants (n = 4). The scale bar represents 500 µm. The data are presented as the means ± SDs; ^§P < 0.05 vs. Phgdh^chon+-complete medium, °P < 0.05 vs. Phgdh^chon−complete medium, ^#P < 0.05 (ANOVA)