Extended Data Fig. 5 |. EP2 blockade restores AKT–GSK3β–GYS1 signalling, glycolysis and mitochondrial respiration to youthful levels.

Data are mean ± s.e.m. unless otherwise specified. a, Quantification of PGE₂ levels in young (below 35 years) and aged (over 65 years) human MDMs treated with or without C52 (100 nM, 20 h). Effects of age and treatment P < 0.0001 by two-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 5 donors per group). b, c, Representative immunoblots (b) and quantification (c) of effects of C52 treatment (100 nM, 20 h) in human MDMs from young (below 35 years) and aged (over 65 years) donors. Two-way ANOVA, age and treatment, P < 0.0001; Tukey’s post hoc test **P = 0.002, ***P = 0.0005, ****P < 0.0001 (n = 6 donors per group). d, Real-time changes in OCR from three independent experiments of young and aged human MDMs treated with or without C52 (100 nM, 20 h; n = 5 donors per group). e, Representative immunoblots of effects of EP2 inhibition on mitochondrial protein levels in young and aged human MDMs (n = 6 donors per group; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane). See Fig. 3h for quantification. f, Membrane potential (TMRE) in young and aged human MDMs treated with or without C52 (100 nM, 20 h). Two-way ANOVA: age P = 0.0009 and treatment P = 0.0333; Tukey’s post hoc test, ***P = 0.0006, **P = 0.0088 (n = 6 donors per group). g, ROS in young and aged human MDMs treated with or without C52 (100 nM, 20 h). Two-way ANOVA: age P = 0.0022 and treatment P = 0.0055; Tukey’s post hoc test **P = 0.0045, ^##P = 0.0084 (n = 7 donors per group). h, Young and aged human MDMs were incubated with U-¹³C-glucose for 20 h with or without C52. Aged human MDMs showed increased labelling in glycogen precursors (G1P and UDP-glucose) and decreased labelling in TCA cycle intermediates. This was prevented with EP2 inhibition; n = 3 donors per group. i, Quantification of TCA cycle metabolites from Fig. 3i. Note normalization of citrate, α-ketoglutarate (α-KG), succinate, fumarate and malate in aged human MDMs with EP2 inhibition. Also note that itaconate, which is increased in models of acute macrophage stimulation with LPS, is not changed with ageing (n = 3 donors per group). Two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001. j, k, Representative traces of real-time changes in OCR (j) and quantification of basal respiration and ECAR (k) from two independent experiments in peritoneal macrophages isolated from young (3–4 months) and aged (18–20 months) mice treated with COX-2 inhibitor SC236 (100 nM, 20 h). Two-way ANOVA: age and treatment, P < 0.0001 and P = 0.004, respectively; Tukey’s post hoc test, ****P < 0.0001 (n = 5 mice per group).