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. 2022 May 2;132(9):e153436. doi: 10.1172/JCI153436

Figure 4. PHGDH inhibition impairs B cell proliferation and de novo serine and glycine synthesis.

Figure 4

(A) Mass isotopologue distribution of U-[13C6]-glucose–derived serine and glycine in B220+ B cells isolated from Phgdh+/+;Cd19-Cre (WT) or Phgdhfl/fl;Cd19-Cre (F/F) or WT C57BL/6J mice. Isolated cells were stimulated with anti-IgM/G antibody, CD40L, and IL-4 for 48 hours and were then cultured for 2 hours in serine/glycine-deplete media with U-[13C]-glucose (and treated with/without PH-755 for WT B cells). (B and C) Representative proliferation profiles and quantification of B220+ B cells from either Phgdh+/+;Cd19-Cre (WT) or Phgdhfl/fl;Cd19-Cre (F/F) mice. Cells were labeled with the Cell Proliferation Dye eFluor 670 and then cultured for 3 days with anti-IgM/G antibody, CD40L, and IL-4 in complete media, serine/glycine-free medium, or serine/glycine-free medium containing glycine and formate (n = 4 per genotype). (D) Cell-cycle analysis of B220+ cells isolated from Phgdh+/+;Cd19-Cre (WT) or Phgdhfl/fl;Cd19-Cre (F/F) mice. Cells were cultured for 48 hours with anti-IgM/G antibody, CD40L, and IL-4 in complete media, serine/glycine-free medium, or serine/glycine-free medium containing glycine and formate; they then underwent BrdU labeling and 7-AAD staining to assess cell cycle. (E and F) Representative proliferation profiles and quantification of B220+ B cells from WT mice. Cells were labeled with the Cell Proliferation Dye eFluor 670 and were then cultured for 3 days with anti-IgM/G antibody, CD40L, and IL-4 in complete media, serine/glycine-free medium, or serine/glycine-free medium containing glycine and formate in combination with DMSO or 10 μM PH-755 (n = 5 per group). (G) Cell-cycle analysis of B220+ B from WT mice. Cells were cultured for 48 hours with anti-IgM/G, CD40L, and IL-4 in complete medium, serine/glycine-free medium, or serine/glycine-free medium containing glycine and formate in combination with DMSO or 10 μM PH-755 before cell-cycle analysis. (H) Activate Caspase-3 on B220+ B cells from either Phgdh+/+;Cd19-Cre (WT) or Phgdhfl/fl;Cd19-Cre (left panel; n = 4 per group) and treated as described in D, and on B cells isolated from WT mice (right panel; n = 3 per group) and treated as described in G. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, by 1-way ANOVA (C and F).