Journal: Cell Reports

Article Title: Inverse Data-Driven Modeling and Multiomics Analysis Reveals Phgdh as a Metabolic Checkpoint of Macrophage Polarization and Proliferation

doi: 10.1016/j.celrep.2020.01.011

Figure Lengend Snippet: Phgdh Expression and Activity Is a Signature of M2 Macrophages (A and B) Total RNA was isolated from unstimulated (Ctrl), LPS-stimulated (100 ng/mL), or IL-4-stimulated (10 ng/mL) wild-type BMDMs after incubation for 24 h (A) or 0, 8, and 16 h (B). Expression levels of the indicated mRNAs were determined by qRT-PCR and normalized to β-actin. Gene expression is presented as fold change versus unstimulated cells. Data are presented as mean ± SEM (n = 4). (C) BMDMs were stimulated for 24 h with IL-4 (10 ng/mL) or solvent control. Whole-cell lysates were analyzed by immunoblotting using the indicated antibodies. Data are representative of 3 independent experiments. (D) Phgdh enzyme activity was determined in protein lysates from wild-type BMDMs stimulated for 24 h with LPS (100 ng/mL) or IL-4 (10 ng/mL) in RPMI-1640 medium (Teknova) with or without glucose, serine and glycine. Phgdh activity was measured by the formation of NADH over time in a buffer containing NAD and the substrate 3-phospho-D-glycerate. Data are presented as mean ± SEM (n = 3–4). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Phgdh Is Required for M2 Polarization of Macrophages (A) Phgdh enzyme activity was determined in protein lysates from wild-type BMDMs stimulated for 24 h with IL-4 (10 ng/mL) in RPMI-1640 medium (Teknova) supplemented with glucose, serine, and glycine.

Techniques: Expressing, Activity Assay, Isolation, Incubation, Quantitative RT-PCR, Solvent, Western Blot

Journal: Cell Reports

Article Title: Inverse Data-Driven Modeling and Multiomics Analysis Reveals Phgdh as a Metabolic Checkpoint of Macrophage Polarization and Proliferation

doi: 10.1016/j.celrep.2020.01.011

Figure Lengend Snippet: Phgdh Is Required for M2 Polarization of Macrophages (A) Phgdh enzyme activity was determined in protein lysates from wild-type BMDMs stimulated for 24 h with IL-4 (10 ng/mL) in RPMI-1640 medium (Teknova) supplemented with glucose, serine, and glycine. Phgdh activity was measured by the formation of NADH over time in a reaction buffer containing NAD and the substrate 3-phospho-D-glycerate. To analyze the activity of the Phgdh inhibitors, 30 μM CBR-5884, 25 μM NCT-503, or solvent control (Ctrl) were added directly to the reaction buffer immediately prior to starting the activity assay. Data are presented as mean ± SEM (n = 4). (B and C) Total RNA was isolated from unstimulated wild-type BMDMs or wild-type BMDMs stimulated for 24 h with IL-4 (10 ng/mL) in the presence or absence of CBR-5884 (30 μM) or NCT-503 (25 μM). Gene expression levels of the indicated mRNAs were determined by qRT-PCR and normalized to β-actin. Inhibition of PHGDH reduced the expression of M2 signature genes Arg1 , Retnla , and Chil3 . Gene expression is presented as fold change versus untreated cells. Data are presented as mean ± SEM (n = 4). (D–F) IL-10 (D), TNF-α (E), and IL-1β (F) production by unstimulated wild-type BMDMs (Ctrl), or wild-type BMDMs stimulated for 24 h with LPS (100 ng/mL) in the presence or absence of CBR-5884 (30 μM), was determined in RPMI-1640 medium (Teknova) with or without glucose, serine, and glycine. Data are presented as mean ± SEM (n = 4). ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Phgdh Is Required for M2 Polarization of Macrophages (A) Phgdh enzyme activity was determined in protein lysates from wild-type BMDMs stimulated for 24 h with IL-4 (10 ng/mL) in RPMI-1640 medium (Teknova) supplemented with glucose, serine, and glycine.

Techniques: Activity Assay, Solvent, Isolation, Expressing, Quantitative RT-PCR, Inhibition

Journal: Cell Reports

Article Title: Inverse Data-Driven Modeling and Multiomics Analysis Reveals Phgdh as a Metabolic Checkpoint of Macrophage Polarization and Proliferation

doi: 10.1016/j.celrep.2020.01.011

Figure Lengend Snippet: M2 Polarization of Macrophages Is Independent of Serine and Glycine in the Medium Total RNA was isolated from unstimulated (Ctrl) or IL-4-stimulated (10 ng/mL) wild-type BMDMs after incubation for 24 h in RPMI-1640 medium (Teknova) with or without glucose, serine, and glycine. mRNA expression levels of Arg1 (A), Retnla (B), Chil3 (C), and Igf1 (D) were determined by qRT-PCR and normalized to β-actin. Gene expression is presented as fold change versus unstimulated cells. Data are presented as mean ± SEM (n = 4).

Article Snippet: Phgdh Is Required for M2 Polarization of Macrophages (A) Phgdh enzyme activity was determined in protein lysates from wild-type BMDMs stimulated for 24 h with IL-4 (10 ng/mL) in RPMI-1640 medium (Teknova) supplemented with glucose, serine, and glycine.

Techniques: Isolation, Incubation, Expressing, Quantitative RT-PCR