Extended Data Fig. 1: CNF1 triggers Caspase-1 activation and ASC specks formation.
a, BMDMs isolated from BALB/c mice were either treated with vehicle (control) or CNF1 (500 ng ml–1), inactive catalytic mutant CNF1 C866S (500 ng/mL) for 6 h, or Nigericin (5 μM) for 30 min. Active Caspase-1 was revealed with FAM-FLICA (green), ASC was stained using an anti-ASC antibody (Texas Red), nuclei and actin filament were stained with Hoechst and phalloidin-Alexa 647 respectively. Cells were analyzed by confocal microscopy. Arrows indicates FAM-FLICA dots that colocalize with the ASC staining. Scale bar: 10μm. b, BMDM isolated from C57BL/6 J mice constitutively expressing ASC-citrine fusion protein (R26-CAG-ASC-citrine) were transfected with the indicated siRNA for 72 h prior to 6 h of CNF1 treatment (500 ng/mL) or treated with vehicle (control). Percent of cells with ASC specks. Data are expressed as the mean ± SEM. Each dot represents 105 cells (n = 2 biologically independent samples). c, BMDM isolated from C57BL/6 J mice constitutively expressing ASC-citrine fusion protein (R26-CAG-ASC-citrine) were treated 6 h with CNF1 (500 ng/mL) or Nigericin (5 μM) for 30 min or vehicle (control). Cells were analyzed for ASC speck formation by flow cytometry as indicated, doublets were excluded using SSC-A and SSC-H plot, cells with a high expression of ASC-citrine were gated and then analyzed for ASC-citrine area (ASC-citrine-A) and ASC-citrine height (ASC-citrine-H). Cells with ASC specks are defined with a higher ASC-H:ASC-A ratio. Experiments were repeated at least three times, and representative data are shown.
