Fig. 2
Enteropathogenic E. coli-mediated inflammasome activation. a WT or WAS KO BMDCs were co-cultured with WT E2348/69 EPEC strain at an MOI of 5 for 3 or 5 h. After co-culture, cells were treated with 100 µg/ml gentamicin for 2 h to kill extracellular bacteria (total 5 or 7 hpi). In parallel experiments, BMDCs were primed with LPS (100 ng/ml) for 3 h prior to infection. After infection, IL-1β protein levels were quantified in culture supernatants by ELISA. Results are mean protein concentration ± SEM of three independent experiments in duplicate. Two-sided Student’s t-test. b WT or WAS KO BMDCs were primed with LPS (100 ng/ml) for 3 h prior to co-culture with WT EPEC (MOI of 5) for 3 h. After fixation, cells were stained for bacteria (DAPI (4′,6-diamidino-2-phenylindole)), ASC (green) and actin phalloidin (red). Images were taken by confocal microscopy (×40 magnification) and the percentage of BMDCs containing ASC specks were quantified. Results are represented as mean ± SEM from three independent experiments. A minimum of 200 BMDCs were evaluated per quantification. Two-sided Student’s t-test. Representative confocal images are shown in Supplementary Fig. 2A. c WT or WAS KO BMDCs were primed with LPS (100 ng/ml) for 3 h, followed by co-culture with WT EPEC (MOI of 5) for 3 h. Intracellular active caspase-1 was detected by FAM-YVAD-FMK (FAM-FLICA) binding, dead cells were stained with PI. The percentage of FLICA and PI double-positive BMDCs were enumerated by flow cytometry (FLICA collected in FL-1 gate; PI collected in FL-2 gate). Results are mean ± SEM of three independent experiments. Two-sided Student’s t-test. A representative flow plot is shown in Supplementary Fig. 2B. d WT or WAS KO BMDCs were co-cultured with WT EPEC (MOI of 5). Three hours after infection, pyroptotic cell death was measured by LDH release, expressed as a percentage of maximum LDH release measured from control, uninfected cells. Results are mean ± SEM of three independent experiments. Two-sided Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001
