Extended Data Fig. 4: NLRP3 activators disrupt EECS inducing actin comets propelling early endosomes.

a, Airyscan images of HeLa cells loaded with 5 µg ml⁻¹ Alexa633-conjugated Transferrin (Tf). Cells were treated with vehicle (Ctrl), 15 µM nigericin or 45 µg ml⁻¹ CL097 for 60 min. Cells were stained with Alexa546-conjugated Phalloidin. Magnifications of areas in dashed squares shown in the lower left corner. Arrowheads indicate co-localization of phalloidin and Tf. Scale bar: 10 µm. b, Live-video imaging of HeLa cells expressing LifeAct-RFP and SNX2-eGFP treated with control (top) or 15 µM nigericin for 60 min. Cells were imaged with 6 second intervals for about 10 min. 5 µg ml⁻¹ Alexa633-conjugated Transferrin (Tf) was added to visualize the endosomal compartment. ROIs are indicated with dashed squares and 5 frames of ROIs with individual channels and merged images are shown. Arrowheads indicate persisting polymerized actin. Scale bar: 10 µm. c, Live-video imaging of HeLa cells expressing both LifeAct-RFP and NLRP3-eGFP treated with 15 µM nigericin for 60 min. Cells were imaged with 6 second intervals for about 10 min. 5 µg ml⁻¹ Alexa633-conjugated Transferrin (Tf) was added to visualize the endosomal compartment. ROI is indicated with dashed squares and 5 frames of ROI with individual channels and merged images are shown. Arrows indicate the formation of NLRP3 puncta on TF-loaded endosomes propelled by actin comets. Scale bar: 10 µm. d, Confocal images of NLRP3 KO BMDMs treated with vehicle (Ctrl), 7.5 µM nigericin or 50 µM CL097 for 20 min. After fixation, cells were co-stained with Alexa488-Phalloidin and an antibody against EEA1. DAPI was used to stain the nucleus. Magnifications of areas in dashed squares shown in the lower left corner. Scale bar: 10 μm. Data shown are representative of at least three independent experiments.