Extended Data Figure 3. Treatment with GSDMD-NT reduces bacterial viability, but does not affect the viability of mammalian cells.

a, Antibiotic-free culture supernatants (concentrated fivefold) from transfected HEK293T cells, collected 30 h after transfection, were added to iBMDMs, which were cultured at 37 °C in 200 μl final volume for 6 h before measuring viability by CellTiter-Glo. b, HEK293T cells, transfected with Flag–GSDMD-NT 6 h earlier, were mixed with an equal number of CFSE-labelled untransfected HEK293T cells and incubated for 18 h before assessing cell death by propidium iodide staining and flow cytometry. c, E. coli and S. aureus were untreated or treated with recombinant GSDMD, wild-type or 4A-mutant GSDMD-NT, or GSDMD-CT (200 nM or indicated concentrations) for 20 min before samples were collected and bacterial growth was assessed by monitoring turbidity by optical density (representative experiments, left). The time to reach OD₆₀₀ of 0.05 above background, which is a quantitative measure of the lag in detectable growth because of fewer viable bacteria, was defined as T_threshold (right). The right graph shows the mean ± s.d. of three technical replicates. d, Bacterial viability after 20 min incubation with indicated proteins (200 nM) or isopropanol. Syto-9 enters live and dead bacteria, PI only enters dead bacteria (representative images, left; percent live cells, right). e, Fluorescence microscopy of mCherry-expressing L. monocytogenes incubated with AlexaFluor 488-GSDMD (activated or not with caspase-11) or AlexaFluor488-GSDMD-CT for 30 min at 37 °C. Data shown are representative of results of three independent experiments. Statistical differences are relative to untreated samples; **P < 0.01 (two-tailed t-test). Scale bars, 5 μm.