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. Author manuscript; available in PMC: 2021 Jul 11.
Published in final edited form as: Nat Microbiol. 2021 Jan 11;6(3):401–412. doi: 10.1038/s41564-020-00832-5

Figure 1. CNF1-triggered caspase-1 activation and IL-1ß maturation requires NLRP3.

Figure 1

(a) BMDMs isolated from BALB/c mice were transfected with the indicated siRNA for 72 h prior to 6 h of CNF1 treatment (500 ng/mL). The active Caspase-1 was detected using the FAM-FLICA probe. Cells harboring FAM-FLICA dots were counted positive using Fiji Software. Each dot represents 200 cells (n=1800 cells). Data are expressed as the mean ± SEM. (b-c) BMDMs extracted from wild-type or NLRP3 knock-out C57BL/6J mice were pretreated 45 min or not with MCC950 (1 μM) prior to treatment for 6 h with CNF1 (500 ng/mL), or the CNF1 catalytic inactive mutant CNF1 C866S (500ng/mL) or Nigericin (5 μM). Cells were analyzed by immunofluorescence and confocal imaging. Active Caspase-1 (FAM-FLICA) is shown in green, NLRP3 in red and nuclei in blue. Scale bar 20 μm. (c) Quantification of FAM-FLICA positive cells in wild-type (blue) or NLRP3 knock-out BMDMs (red). Each dot represents 100 cells (n=600 cells). Data are expressed as the mean ± SEM. Statistical analyses were performed using a two-tailed unpaired Student’s t-test. (d) wild-type or NLRP3 knock-out BMDMs were treated with CNF1 (500 ng/mL) and/or LPS (100 ng/mL) for 8 h prior to supernatant and cell lysates collection and immunoblot analysis. The numbers on the side of the immunoblots indicate molecular weight (kDa). Experiments were repeated at least three times, and representative data are shown.