Fig. 1.

PHGDH inhibits M(IFN-γ) but promotes M(IL-4) polarization through its enzymatic activity. Wild-type bone marrow-derived macrophages (BMDMs) were pretreated with the PHGDH inhibitor CBR-5884 (15 μM) for 12 h and were then stimulated with IFN-γ (100 ng/ml) for 12 h (a, b) or with IL-4 (20 ng/ml) for 24 h (c, d), followed by qRT‒PCR or western blot analysis of M1 (a, b) and M2 (c, d) marker expression. Phgdh^fl/flLyz2-Cre^- (PHGDH-WT) and Phgdh^fl/flLyz2-Cre⁺ (PHGDH-KO) mouse BMDMs were stimulated with IFN-γ for 12 h or the indicated times, followed by qRT‒PCR (e) or western blot (f) analysis of M1 marker expression. PHGDH-WT and PHGDH-KO BMDMs were stimulated with IL-4 for 24 h or the indicated times, followed by qRT‒PCR (g) or western blot (h) analysis of M2 marker expression. RAW264.7 cells with siRNA-mediated PHGDH silencing or stable shRNA-mediated PHGDH knockdown were transfected with an RNA interference (RNAi)-resistant PHGDH ectopic expression plasmid or a plasmid expressing a catalytically dead PHGDH mutant (V425M) and were then either stimulated with IFN-γ for 12 h to evaluate M1 marker expression by qRT‒PCR (i) or western blot analysis (j) or stimulated with IL-4 for 24 h to detect M2 marker expression by qRT‒PCR (k) or western blot analysis (l). siCtrl siControl, shCtrl shControl, EV empty vector. The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 3) (a, c, e, g, i, k) or are representative of three independent experiments (b, d, f, h, j, l). *p < 0.05, **p < 0.01, ***p < 0.001