Fig. 1. Polymerization kinetics of plasma membrane NINJ1.
a,b, Western blot analysis of endogenous GSDMD and NINJ1 in primed BMDMs after nigericin stimulation (Nig) for 1.5 h (a) or for 2, 5, 15, 25, 55 or 85 min (b) followed by treatment with the membrane-impermeable BS3 crosslinker for 5 min. FL, full length. Of note, tubulin, used here as a loading control, is crosslinked owing to BS3 entry through GSDMD pores under nigericin-treated conditions. Time in b is the total incubation time for nigericin and BS3 treatment. Short exp., short exposure. For gel source data, see Supplementary Fig. 1. c, LDH release from primed BMDMs after nigericin stimulation. d, Time-lapse fluorescence confocal microscopy of HeLa cells co-expressing hNINJ1–GFP and opto-casp1 following photo-activation. Images show the NINJ1–GFP fluorescence at the basal plane of the cell and the influx of DRAQ7 (maximum (max.) projection from a z-stack) to track plasma membrane permeabilization. Time was normalized to the onset of increase in DRAQ7 nuclear fluorescence. White arrows indicate regions that are enlarged in the insets. Scale bar, 10 µm. e–g, Normalized quantification of the distribution inhomogeneity of NINJ1–GFP (e), HATMD–GFP (f) or E-cadherin–GFP (g) at the basal plane of cells, and DRAQ7 nuclear fluorescence intensity after photoactivation of opto-casp1 (F). The distribution inhomogeneity at each time point (Dt) was normalized to the distribution inhomogeneity at the initial time point of the experiment (Di). Data are mean ± s.d. Data are representative of 2 (b), 3 (a) or 14 (d) independent experiments, or pooled from 2 independent experiments performed in triplicate (c) or at least 10 (e–g) independent experiments.