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. 2022 Sep 30;19(11):1263–1278. doi: 10.1038/s41423-022-00925-7

Fig. 6.

Fig. 6

Serine metabolism reduces IGF1 expression by increasing the promoter abundance of SAM-dependent H3K27me3. qPCR analysis of Igf1 mRNA expression (a) and ELISA (b) of IGF1 in the supernatants of PHGDH-WT and PHGDH-KO BMDMs. qPCR analysis of Igf1 mRNA expression (c) and ELISA (d) of IGF1 in the supernatants of RAW264.7 cells transfected with empty vector or the PHGDH overexpression plasmid. qPCR analysis of Igf1 mRNA expression (e) or ELISA (f) of IGF1 in the supernatants of BMDMs starved of SG. g Analysis of Igf1-Luc activity in PHGDH-WT and PHGDH-KO BMDMs starved of SG. h qPCR analysis of Igf1 mRNA expression in PHGDH-WT and PHGDH-KO BMDMs supplemented with or without 400 μM SAM. i Analysis of Igf1-Luc activity in RAW264.7 cells transfected with siControl or siPHGDH and supplemented with or without 400 μM SAM. j, k Western blot analysis of histone modification markers in PHGDH-WT and PHGDH-KO BMDMs supplemented with or without 400 μM SAM for 12 h. l ChIP‒qPCR analysis of H3K27me3 enrichment at the indicated positions in the Igf1 promoter in PHGDH-WT and PHGDH-KO BMDMs. PHGDH-WT and PHGDH-KO BMDMs were supplemented with or without 400 μM serine or 400 μM SAM for 12 h and were then treated with or without IFN-γ for 12 h (m–o) or IL-4 for 24 h (p, q), followed by ChIP‒qPCR analysis of H3K27me3 enrichment in the promoter of the Igf1 gene. r, s qPCR analysis of Jmjd3 and Ezh2 mRNA expression in PHGDH-WT and PHGDH-KO BMDMs. t Western blot analysis of EZH2 expression in PHGDH-WT and PHGDH-KO BMDMs. BMDMs with siRNA-mediated EZH2 silencing were transfected with empty vector or the PHGDH expression plasmid. qPCR analysis of Igf1 mRNA expression (u), western blot analysis performed with the indicated antibodies (v), and ChIP‒qPCR analysis of H3K27me3 enrichment in the promoter of the Igf1 gene (w). The data are from three independent experiments with biological duplicates in each and are shown as the mean ± SEM values (n = 3) (a–i, l–s, u, w) or are representative of three independent experiments (j, k, t, v). NS not significant (p ≥ 0.05); *p < 0.05; **p < 0.01; ***p < 0.001

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