Extended Data Fig. 2. NINJ1 quickly polymerizes at the plasma membrane upon pyroptosis activation.
a,b, Schematic representation of the experimental setup used to analyze NINJ1 polymerization at the plasma membrane upon optogenetic induction of GSDMD-driven pyroptosis in single cells: HeLa cells expressing Opto-Casp1 (red dots) are photo-activated with 488 nm light to induce rapid Opto-Casp1 clustering and caspase activation, triggering GSDMD pore formation and influx of DRAQ7 (a membrane-impermeable DNA dye) (a). Time-lapse fluorescence confocal microscopy allows to follow NINJ1-GFP localization and clustering in different Z-planes of the cell (b). c—e, Time-lapse fluorescence confocal microscopy images (basal or central plane of cells) of HeLa cells co-expressing Opto-Casp1 and hNINJ1-GFP (c), HATMD-GFP (d) or E-cadherin-GFP (e), after photo-activation. DRAQ7 influx (maximum projection from a Z-stack) shows plasma membrane permeabilization during cell death. Time was normalized to the onset of increase in DRAQ7 nuclear fluorescence. Scale bars correspond to 10 µm and white arrows point to inset images. Data are representative from fourteen (c), eleven (d) or ten (e) independent experiments.