Extended Data Fig. 2: NLRP3 inflammasome activation by CNF1 does not induce pyroptosis.
a, BMDMs isolated from C57BL/6 J mice were treated with vehicle (control), Nigericin (5 μM) or CNF1 (500 ng/mL or 5 μg/mL) with or without LPS (100 ng/mL). Propidium iodide (PI) uptake was monitored over time (red object count) by real time imaging. Data are expressed as mean ± SD. 104 cells were analyzed for each replicate (n = 4 independent wells). b, BMDMs isolated from C57BL/6 J mice were treated with vehicle (control, n = 6 independent experiments), LPS (100 ng/mL, n = 4 independent experiments), LPS and CNF1 (500 ng/mL, n = 6 independent experiments) or LPS and Nigericin (5 μM, n = 4 independent experiments), and LDH release was assessed. Data are expressed as the mean ± SEM. Statistical analyses were performed using a two-tailed nonparametric Mann Whitney test. c, BMDMs isolated from C57BL/6 J mice were treated either with Nigericin (5 μM) for 30 min or CNF1 (0.5, 1 or 5 μg/mL) for 8 h and GSDMD cleavage in cell lysates is shown. d, BMDMs isolated from C57BL/6 J wild-type or CASP1, CASP11, PYCARD (coding for ASC) or GSDMD knock-out mice were untreated or treated with CNF1 (500 ng/mL) for 6 h and were analyzed for Caspase-1 activation using the FAM-FLICA probe. Data are expressed as the mean ± SEM. Statistical analyses were performed using a two-tailed unpaired Student’s t-test. Each dot represents 100 cells (n = 700 cells). e, BMDMs isolated from wild-type or GSDMD knock-out mice were treated with CNF1 (500 ng/mL) and LPS (100 ng/mL) for 8 h as indicated. Supernatants and cell lysates were analyzed by immunoblot. The numbers on the side of the immunoblots indicate molecular weight (kDa). Experiments were repeated at least three times, and representative data are shown.
