Fig. 4 |. Preferential IL-1β release from macrophages.

a, b, Preferential release of mature IL-1β from glycine-protected living iBMDMs permeabilized by GSDMD, shown by immunoblotting (a) and LDH release (n = 3 biological replicates) (b). UT: Untreated. N: Nigericin. G: Glycine. Uppercase H: High dose at 20 μM. Sup: Supernatant. WCL: Whole cell lysate. Pro: Precursor. Mat: Mature. c, Comparison of IL-1β release across GSDMD-KO iBMDMs expressing with WT or AP-mutant GSDMD, and across pro-IL-1β-KO iBMDMs expressing WT or AP’-mutant pro-IL-1β. EV: Empty vector, a mock transduction control. d-f, Release of mature IL-1β from living iBMDMs without glycine protection, characterized by immunoblotting (d), LDH release (n = 3 biological replicates) (e), and ELISA (n = 3 biological replicates) (f). Uppercase L: Low dose at 0.5 μM. g, h, Comparison of IL-1β release across GSDMD-KO iBMDMs expressing WT or AP-mutant GSDMD without glycine protection, evaluated by immunoblotting (g) and ELISA (n = 3 biological replicates) (h). i, j, Comparable leakage of pro-IL-1β and mature IL-1β from GSDMD-KO iBMDMs perforated by SLO and PFO (i) at cytotoxic and non-toxic concentrations shown by ATP-based cell death (n = 3 biological replicates) (j). Uppercase H: High dose at 625 nM. Uppercase L: Low dose at 0.16 nM. k, Schematic diagram for GSDMD pore formation and IL-1 release. The question mark indicates other possible assembly mechanisms. Data shown in b, e, f, h, and j are mean ± s.d.. Data shown in a and d are representative of three, and data shown in c, g, and i two, independent experiments.