Extended Data Fig. 5: Kinetics of noncanonical activation of the NLPR3 inflammasome in macrophages in response to virulent and avirulent bacteria.

a, LDH released in culture supernatants of macrophages at indicated time points post-stimulation with Live E. coli (avirulent), virulent E. coli or virulent Salmonella. LDH measured by cytotoxicity assay. b,c, Kinetics of SYTOX Red incorporation over 72 h in macrophages stimulated with Live E. coli or virulent E. coli, Salmonella ΔSpi1/2 (avirulent) or wild type (WT) (virulent), or Shigella BS103 (avirulent) or WT (virulent) (b), and representative images of SYTOX Red incorporation at selected time points (c). Peaks of SYTOX incorporation occurred faster (blue boxes) in response to virulent bacteria, before decreasing likely due to destruction of cell structure, while macrophages stimulated with avirulent bacteria were still incorporating SYTOX and peaked later (orange boxes). Scale bar=150 µm. d, Counting of live macrophages at the indicated time points post stimulation with Live E. coli or virulent E. coli. Counts were normalized to the unstimulated macrophages for each time point. e, Immunoblots of macrophage WCE at the indicated time points post stimulation with Live E. coli, virulent E. coli or virulent Salmonella. f, Immunoblots of macrophage concentrated supernatants 6 h post stimulation with Live E. coli or virulent E. coli. Error bars, mean ± s.e.m. Bacteria:macrophage =20:1 for E. coli and virulent E. coli, 5:1 for Salmonella and Shigella strains. Results represent at least three independent experiments.