Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture

Cell detachment and blebbing during necroptosis and pyroptosis. a B6Nlrp1b+ BMDMs were stimulated with TNF+BV6+zVAD-fmk (TBz: 20 ng/ml, 2 μM and 50 μM, respectively) and imaged in culture media containing Sytox Green. b The rCTB-stained B6Nlrp1b+ BMDMs were stimulated with LeTx and imaged as in (a). a, b Confocal images were acquired every 3？min. c, d B6Nlrp1b+ BMDMs pretreated with Y27632 (10？μM) (c) or (？)-blebbistatin (10？μM) (d) were stimulated with LeTx or TBz and imaged. Fluorescent micrographs show the maximum intensity projection (Sytox Green) or the single plane (rCTB) of a representative cell from 3 independent experiments (TBz n？=？30; LeTx n？=？30; LeTx+Y27632 n？=？25; TBz+Y27632 n？=？23; LeTx+(？)-blebbistatin n？=？19; TBz+(？)-blebbistatin n？=？20). In all panels, time point zero indicates the first detection of Sytox Green. All scale bars, 10？μ