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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Nature. 2021 Jan 20;590(7844):122–128. doi: 10.1038/s41586-020-03160-0

Fig. 3 |. EP2-directed glycogen synthesis regulates macrophage bioenergetics and immune responses.

Fig. 3 |

Human MDMs were derived from young (below 35 years of age) and aged (over 65 years of age) donors. Data are mean ± s.e.m. unless otherwise specified. a, Basal respiration and ECAR in human MDMs transfected with short hairpin RNA (shRNA) against GYS1 (shGYS1) or with control scrambled shRNA; three independent experiments. Two-way ANOVA, age and genotype P < 0.0001; Tukey’s post hoc test *P = 0.0225, **P = 0.0075, ***P < 0.001 (n = 6 donors per group). b, Hierarchical clustering of significantly regulated immune factors in human MDMs 20h after transfection of shGYS1 or scrambled shRNA (scr) (n = 5 young, n = 6 aged donors). c, Mean fluorescence intensity (MFI) of three independent flow cytometry experiments in young and aged human MDMs transfected with shGYS1 or with control scrambled shRNA. Two-way ANOVA, age and shRNA P < 0.0001; Tukey’s post hoc test, *P = 0.0178, **P = 0.0041, #P = 0.0003, ***P = 0.0006, ****P < 0.0001 (n = 20,000–40,000 cells per point; n = 3 donors per group). d, Basal respiration and ECAR of young and aged human MDMs transfected with shGYS1 or control scrambled shRNA and stimulated 8 h later with vehicle or butaprost (but; 100 nM, 20 h). Two-tailed Student’s t-test, **P = 0.0018, ****P < 0.0001 (n = 4 donors per group). e, Hierarchical clustering of significantly regulated immune factors in human MDMs from d (n = 5 donors per age group). f, Glycogen levels in young and aged human MDMs treated with vehicle or C52 (100 nM, 20 h). Two-way ANOVA, age and treatment P < 0.0001; Tukey’s post hoc test, ****P < 0.0001 (n = 6 donors per group). g, Basal respiration and ECAR in young and aged human MDMs treated with vehicle or C52 (100 nM, 20 h). Two-way ANOVA, age and treatment P < 0.0001; Tukey’s post hoc test, ****P < 0.0001 (n = 5 donors per group). h, Levels of mitochondrial proteins quantified by immunoblot in young and aged human MDMs treated with vehicle or C52 (100 nM, 20 h). Two-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (n = 6 donors per group). i, Liquid chromatography coupled to mass spectrometry (LC–MS) measurements of glycolytic and TCA cycle metabolites in young and aged human MDMs treated with vehicle or C52 (100 nM, 20 h; n = 3 donors per group). j, Phagocytosis of fluorescent E. coli particles in young and aged human MDMs treated with vehicle or C52 (100 nM, 20 h). Two-way ANOVA with Tukey’s post hoc test, ***P = 0.0006, ****P < 0.0001 (n = 6 donors per group).