Extended Data Fig. 4 |. Knockdown of GYS1 promotes macrophage glucose metabolism and anti-inflammatory polarization.

Data are mean ± s.e.m. unless otherwise specified. a–f, Human MDMs are from donors (age (mean ± s.e.m.) 47.2 ± 1.582 years); g–i, Human MDMs are from young (below 35 years) and aged (over 65 years) donors. a, Representative immunoblots and quantification of human MDMs transfected with two different shRNAs to human GYS1 at 8 h. P < 0.0001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 6 donors per group). b, Quantification of glycogen levels in human MDMs transfected with shRNAs to GYS1 at 8 h. P < 0.0001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 6 donors per group). c, Representative traces and quantification of OCR and ECAR for three independent experiments in human MDMs transfected with shRNAs for GYS1 at 8 h (n = 5 donors per group). P < 0.0001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 6 donors per group). Black arrows represent addition of oligomycin (1 μM), FCCP (2 μM), and rotenone/antimycin (500 nM), respectively, at time points indicated. d, Hierarchical clustering of targeted metabolomics for glycolysis, pentose phosphate shunt and TCA cycle metabolites in human MDMs transfected with shRNA to GYS1 at 8h (n = 5 donors per group). e, Isotope tracing of U-¹³C-glucose in human MDMs transfected with shRNA to GYS1 at 8 h reveals a decreased labelling in the glycogen precursor UDP-glucose and an increase in glycolytic intermediates F-1,6-BP and pyruvate (n = 6 donors per group). f, Representative flow cytometry histograms of three independent experiments for the pro-inflammatory markers CD86 and CD64 and anti-inflammatory markers CD206 and CD163 in human MDMs treated with or without shRNA to GYS1. Bottom, quantification of MFI, two-tailed Student’s t-test, *P < 0.05, **P < 0.01 (n = 20,000–40,000 cells per point, n = 3 donors per group). g, Representative trace of real-time changes in OCR from three independent experiments of young (below 35 years) and aged (over 65 years) human MDMs treated with or without shRNA to GYS1 (n = 5 young, n = 6 aged donors). h, Representative histograms of anti- and pro-inflammatory surface markers in young and aged human MDMs treated with or without shRNA to GYS1 (n = 3 donors per group). i, Representative traces of real-time changes in OCR from two independent experiments in young (below 35 years) and aged (over 65 years) human MDMs transfected with shRNA to GYS1 and treated 8 h later with butaprost (100 nm) for 20 h. OCR in GYS1-deficient aged human MDMs does not change with activation of the EP2 receptor by butaprost (n = 4 donors per group).