Extended Data Fig. 1: Inflammasomes trigger the eruption of F-actin projections in macrophages.

a-b, Live fast Airyscan confocal imaging of macrophages from mice expressing the LifeAct- peptide fused to eGFP (green) a, primed for 4 h with 100 ng ml⁻¹ ultrapure E. coli LPS and stimulated with 5 µM nigericin, or b, primed for 16 h with 1 µg ml⁻¹ PAM3CSK4 and then transfected with 2 µg ml⁻¹ ultrapure E. coli LPS delivered using FuGENE HD, for the time indicated in each panel. Macrophages were incubated with 150 nM Sytox-red (magenta) during imaging. LifeAct-eGFP insets are displayed in grey within each panel. c-d, Airyscan confocal imaging of fixed macrophages labelled with DAPI nuclear stain (grey), phalloidin F-actin stain (green, and grey inverted insets in the lower right of panels), and α-ASC antibody (magenta, circular inset). Macrophages were c, unstimulated, primed with LPS, or LPS-primed for 4 h and stimulated for 30 min with nigericin, or d, mock transfected with FuGENE HD with or without prior priming with PAM3CSK4 for 16 h, or PAM3CSK4-primed and stimulated for 2 h with intracellular LPS packaged with FuGENE HD. a-d, Images are maximum intensity projections of Z-stack acquisitions, and representative of n = 3 independent biological experiments. e, Confocal imaging of fixed macrophages labelled with phalloidin (green), DAPI (grey), and α-ASC antibody (magenta). Macrophages were primed with LPS for 4 h and stimulated with 1.25 mM ATP for 1 h, or 5 µM nigericin for 30 min, in the presence or absence of 10 µM MCC950 added 30 min before ATP or nigericin. Unprimed macrophages were stimulated with 250 ng ml⁻¹ PA and 125 ng ml⁻¹ Fla1-LFn (PA-FlaTox) for 1 h to induce NLRC4 signalling. Images are maximum intensity projections of Z-stack acquisitions. f, LDH release of e, plotted as mean of technical duplicates. g, Airyscan confocal imaging of pyroptotic projections labelled with phalloidin (green) and α-MYO10 antibody (magenta). Plots of mean fluorescence intensity (MFI) in regions outlined by dashed yellow rectangles (upper) are displayed in lower panels. Primed macrophages were stimulated with cytosolic LPS, as described earlier. Images are single Z-planes and representative of n = 3 independent biological experiments. h, Fixed Airyscan confocal imaging of human monocyte-derived macrophages (HMDM), labelled with F-actin stain phalloidin (green), MYO10 (magenta) and DAPI (grey). HMDM were LPS primed or co-administered LPS plus nigericin (10 µM), or 250 ng ml⁻¹ PA plus 20 ng ml⁻¹ PrgI-LFn (PA-PrgI) to induce NLRC4 signalling. Images are maximum intensity projections of Z-stack acquisitions and representative of n = 3 independent biological experiments. All scale bars = 10 µm except where indicated.