Figure 1.
Figure 1. The pyroptotic cell corpse consists of insoluble cellular contents. (A) WT BMMs were infected at an MOI of 20–25 for 2.5 h with L. monocytogenes, C. rodentium, B. thailandensis, or SPI1-repressed S. Typhimurium (under conditions where minimal inflammasome detection is observed) or with SPI-1–induced S. Typhimurium (which activates the NLRC4 inflammasome). Cell lysis was determined by LDH release. (B) WT BMMs were treated with PBS or 3 µg/ml FlaTox for 2 h. Percentage cell death was determined by counting based on morphology in DIC microscopy, which was equivalent to counting based on PI positivity (not depicted; C–F and K). BMMs were infected with SPI1-induced WT S. Typhimurium for 2.5 h (E) or treated with 3 µg/ml FlaTox for 2 h (C, D, F, and K), and then imaged by confocal microscopy. MitoTracker, Bodipy, WGA, dextran, and GFP labels were endogenous or added before treatment, and live cells were imaged. For vimentin, tubulin, phalloidin, caspase-1, and DAPI, cells were fixed and stained before imaging. Caspase-1–positive-speck in dead BMMs is indicated by a white arrow (K). Dead BMMs were identified by PI-staining (E and K) or DIC (F). Images are representative; quantitation of cellular markers was done in ImageJ by measuring the total intensity of ∼50 individual live and dead BMMs at time 0 and 2.5 h after infection or 2 h after FlaTox. (D) To determine the number of surface-attached cells before and after pyroptosis, the number of GFP (live) and PI (dead) cells from (C) were counted at time 0 and 2 h after FlaTox. (G–J) Electron micrographs of WT BMMs were infected with SPI1-repressed S. Typhimurium or L. monocytogenes followed by 3 µg/ml FlaTox treatment (G, H, and J) or infected with SPI1-induced S. Typhimurium (I). Frank rupture of the plasma membrane (arrowhead) and mitochondria with collapsed cristae (arrow). Dotted squares show individual bacteria, with greater magnification on right panel. Data are representative of two (A–F and K) and one (G–J) experiments. Error bars represent SE. P-values were determined by a two-tailed Student’s t test; *, P < 0.05.

The pyroptotic cell corpse consists of insoluble cellular contents. (A) WT BMMs were infected at an MOI of 20–25 for 2.5 h with L. monocytogenes, C. rodentium, B. thailandensis, or SPI1-repressed S. Typhimurium (under conditions where minimal inflammasome detection is observed) or with SPI-1–induced S. Typhimurium (which activates the NLRC4 inflammasome). Cell lysis was determined by LDH release. (B) WT BMMs were treated with PBS or 3 µg/ml FlaTox for 2 h. Percentage cell death was determined by counting based on morphology in DIC microscopy, which was equivalent to counting based on PI positivity (not depicted; C–F and K). BMMs were infected with SPI1-induced WT S. Typhimurium for 2.5 h (E) or treated with 3 µg/ml FlaTox for 2 h (C, D, F, and K), and then imaged by confocal microscopy. MitoTracker, Bodipy, WGA, dextran, and GFP labels were endogenous or added before treatment, and live cells were imaged. For vimentin, tubulin, phalloidin, caspase-1, and DAPI, cells were fixed and stained before imaging. Caspase-1–positive-speck in dead BMMs is indicated by a white arrow (K). Dead BMMs were identified by PI-staining (E and K) or DIC (F). Images are representative; quantitation of cellular markers was done in ImageJ by measuring the total intensity of ∼50 individual live and dead BMMs at time 0 and 2.5 h after infection or 2 h after FlaTox. (D) To determine the number of surface-attached cells before and after pyroptosis, the number of GFP (live) and PI (dead) cells from (C) were counted at time 0 and 2 h after FlaTox. (G–J) Electron micrographs of WT BMMs were infected with SPI1-repressed S. Typhimurium or L. monocytogenes followed by 3 µg/ml FlaTox treatment (G, H, and J) or infected with SPI1-induced S. Typhimurium (I). Frank rupture of the plasma membrane (arrowhead) and mitochondria with collapsed cristae (arrow). Dotted squares show individual bacteria, with greater magnification on right panel. Data are representative of two (A–F and K) and one (G–J) experiments. Error bars represent SE. P-values were determined by a two-tailed Student’s t test; *, P < 0.05.

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