Fig. 8.

GSDMD deficiency prevents LPS transfection-induced early Ca²⁺ influx and mitochondrial decay. a, c Pam3csk4-primed BMDMs of the indicated genotypes were preloaded with the cell-permeant Ca²⁺ indicator Fluo4 and imaged after transfection with LPS (2 µg/ml, Fugene+LPS), treated with Fugene alone or 'mock'-treated in culture media containing PI. Confocal images were acquired every 2 min. Fluorescent micrographs show the maximum intensity projection of a representative cell. b, e BMDMs of the indicated genotypes were preloaded with TMRM and imaged after transfection with LPS (2 µg/ml, Fugene+LPS), treated with Fugene alone, or 'mock'-treated in culture media containing Sytox Green. Confocal images were acquired every 3 min. Fluorescent micrographs show the maximum intensity projection of a representative cell. d, f Graphs show the percentage of mean fluorescence intensity (MFI) calculated as described in the Methods section, and values represent the mean ± SD of individual cells imaged in 3 or 4 independent experiments (Fluo4: WT n = 18, GSDMD^-/- n = 28; TMRM: WT n = 18, GSDMD^-/- n = 29). Single-cell plots are shown in Supplemental Figure 14. In all panels, time point zero indicates the first detection of PI/Sytox Green. All scale bars, 10 µm