Extended Data Fig. 2. S100A8/A9 release is dependent on NLRP3 inflammasome.
a Amount of processed caspase-1 (casp-1 p20/casp-1 p22) in lysates of human neutrophils was determined and normalized to GAPDH (n = 5 independent experiments). b-c Caspase-1 activity was determined in FLICA dye-loaded, human neutrophils pretreated with VX-765 (10 µM) upon stimulation with E-selectin or PBS (control) for 10 min. Cells were analyzed by confocal microscopy. Quantification of b mean FLICA positive area and c overall mean fluorescence intensities (MFI) of FLICA signal/cell (47 (control) and 57 (E-selectin) cells of n = 4 independent experiments). d Representative super-resolution micrographs (maximum projection of 10 planes around cell centers; arrows: GSDMD-NT (green) at the plasma membrane (magenta)) and e Plasma membrane (PM) translocation index (PM vs cytosol GSDMD-NT MFI ratio) of human neutrophils treated with PBS and E-selectin (19 (control) and 19 (E-selectin) cells of n = 4 independent experiments). f WT mice were i.p. injected with NLRP3-inhibitor MCC950 or vehicle control 1 h prior to intrascrotal TNF application. Serum S100A8/A9 levels were analyzed by ELISA before and 2 h after TNF stimulation (n = 4 (WT), 5 (WT + MCC950) mice/group). g Bone marrow neutrophils from WT mice pretreated with MCC950 (1 µM, 30 min) or vehicle control were incubated with E-selectin or PBS. (n = 5 mice/group). S100A8/A9 levels were analyzed in supernatants by ELISA. Rolling velocities of h MCC950 (1 µM, 30 min) or vehicle control (65 control and 68 MCC950 treated cells from n = 3 mice/group) pretreated WT neutrophils were assessed in E-selectin/ICAM-1-coated flow chambers. Rolling velocities were determined in i MCC950 (1 µM, 30 min) or vehicle control (104 (control) and 81 (MCC950) cells from n = 3 independent experiments/group) pretreated human neutrophils. Data are presented as mean ± s.e.m., (two-tailed paired student’s t-test for a-c, e, h and i, two-way ANOVA, Sidak’s multiple comparison for f; two-way RM ANOVA, Sidak’s multiple comparison for g), as representative Western blot for a, as representative micrographs for d and as cumulative distribution for h and i. ns: not significant.