Extended Data Fig. 4: De novo serine synthesis is not conserved in NK cells.

(a) Western blot showing PHGDH protein expression in human (above) or mouse (below) NK cells cultured in Ser+ or Ser- media for 48 h before protein extraction. (b) Schematic of [U-¹³C]-glucose tracing of naïve or 48 h stimulated human and mouse NK cells. (c) Percent ¹³C labelling of indicated nonessential amino acids in human or mouse NK cells cultured with [U-¹³C]-glucose for 24 h with cytokine stimulation immediately after isolation. (d) Isotopologue distribution of labelled serine in naïve or D3 IL-15-stimulated mouse NK cells labelled with [U-¹³C]-glucose in the final 24 h of culture. (e, f) Percent specific lysis of K562 cells (e) and percent IFN-γ⁺ (f) of human NK cells cultured for 48 h in asparagine-replete (Asn+) or asparagine-free (Asn-) media with IL-2/15 for 48 h before stimulation. (g) Immunoblot showing protein expression of PHGDH and β-actin loading control in nontargeting control (NTC) edited or PHGDH^CRISPRa human NK cells 48 h after electroporation. (h) Abundance of ¹³C-labelled serine in NTC or PHGDH^CRISPRa human NK cells. (i) Indel frequency calculated using SYNTHEGO ICE of mouse NK cells edited with sgRosa26 or sgPhgdh cRNP complexes. (j) Relative serine abundance in NTC or Phgdh^cRNP mouse NK cells cultured in Ser+ (left) or Ser- (right) media. (k) Relative phosphoserine abundance in NTC or Phgdh^cRNP mouse NK cells cultured in Ser- media. Data represent mean ± SEM or individual paired donors across at least two independent experiments. Data are representative of (a) n = 3 independent human donors and mice, (c) n = 5 mice and 4 independent donors, (d) n = 5 mice, (e, f) n = 5 independent human donors, (g) n = 3, and (h) n = 7 donors and (j, k) n = 5 mice per group. *p < 0.05, **p < 0.01, ****p < 0.0001 by (c-f) two-way ANOVA, (h) one-way ANOVA, or (j, k) two-sided Student’s t-test. Exact p-values: (c) p = 0.0084, p = 0.0097; (d) p < 0.0001; (h) p = 0.0037 NTC ser+ vs PHGDH^CRISPRa ser-; p = 0.0202 NTC ser- vs PHGDH^CRISPRa ser-; (j) p = 0.0295; (k) p = 0.0311. Created in BioRender. Li, J. (2025) https://BioRender.com/v7pq7fo.