Extended Data Fig. 3: Inflammasome activators induce filopodia under non-lytic conditions, and in non-myeloid cells.

a, Fixed Airyscan confocal imaging of human bronchial epithelial cells (HBEC), labelled with F-actin stain phalloidin (green), Myo10 (magenta) and DAPI (grey). Cells were primed for 16 h with 1 µg ml⁻¹ PAM3CSK4 (PAM), and CASP4 was activated with LPS transfection for 5.5 hours. b-c, Representative immunofluorescence images of BMDMs or e-f, BMDCs primed with LPS and treated with nigericin or PGPC for 0.5 to 6 h to induce pyroptosis or hyperactivation and filopodia formation. b, e, Insets depict filopodia. Cells were labelled with F-actin stain phalloidin (green), ASC (magenta) and DAPI (grey). c, f, Representative Airyscan immunofluorescence images of filopodia in pyroptotic or hyperactive BMDMs or BMDCs (2 h stimulation). Plots of MFI in regions outlined by dashed yellow rectangles are displayed in lower panels. Cells were labelled with F-actin stain phalloidin (green) and Myo10 (magenta). d, g, Quantification of branched projections per cell from BMDMs or BMDCs treated as indicated to induce pyroptosis or sub-lytic inflammasome signalling. Protrusions from 40 randomly chosen cells with filopodia were counted per condition and time point to assess the percentage of projections that were branched. Data were analysed using a Kruskal Wallis test with Dunn’s multiple testing correction (n = 25–72 from three independent biological experiments). All images are maximum intensity projections of Z-stack acquisitions and representative of n = 3 independent biological experiments. All scale bars = 10 µm. Violin plots show mean (solid line) and first and third quartiles (dotted lines). Statistical significance: *** p ≤ 0.001; **** p ≤ 0.0001.