Extended Data Fig. 5: Release of GSDMD-dependent metabolites promotes tissue regeneration.
From: Gasdermin D-mediated metabolic crosstalk promotes tissue repair
a, Schematic of supernatant collection and ultracentrifugation. Macrophages were stimulated with 500 ng/mL LPS for 4 h. Thirty minutes before the second signal (3.5 h post LPS stimulation), 10 mM glycine was supplemented to maintain plasma membrane integrity. The second signals, 10 µM nigericin or 1 µg/mL poly(dA:dT), were added to induce GSDMD pore formation. b-d, Quantification of IL-1β (b), IL-6 (c) and TNFα (d) by ELISA (n = 3). e, f, LDH levels in supernatants determined by LDH releasing assay (n = 4) and PI+ cells detected by flow cytometry (n = 3). g-j, Volcano plot of metabolite levels with indicated comparisons, representing total lytic release (g), glycine-induced secretion (h), active secretion (i), and GSDMD pore-dependent active release (j), related to Fig. 3c,d. k, Schematic of 11,12-EET biogenesis and hydrolysis. l, 11,12-EET levels in supernatants determined by ELISA (n = 2, technical replicates). m, PCA plot of targeted metabolites of cell lysates and supernatants, related to Fig. 3e. n, The proportion of 11,12-EET secretion in the supernatant relative to the intracellular quantity, related to Fig. 3e. o,p, Representative images (o) and quantification (p) of C2C12 cell fusion index and myotube size with/without 11,12-EET treatment (n = 6). Scale bar, 200 μm. q, Schematic of primary MuSC isolation procedure. r,s, Representative scanning electron microscope image (r) and statistics (s) of 11,12-EET treated (n = 30) and control (n = 40) MuSCs. t,u, Representative flow cytometry contour plots, related to Fig l,m. A representative (l,o,p,r) or a pool (b-f, s-u) of at least two independent experiments is shown. Unpaired two-tailed t test applied for (b-d,n,p,s). Two-way ANOVA applied for (e,f) with Šidák correction. Mean ± SEM, except Mean ± SD for 5 l.
