Figure 3.

N-terminal cysteine mutations uncouple speck formation and inflammasome function. (For ASC speck analysis) HEK293T cells were transfected with 50 ng of GFP-ASC with 100 ng NLRP3 or pcDNA3. Eighteen hours post-transfection, cells were either stimulated with nigericin agonists or left untreated, or 4 h after transfection, cells were infected with Francisella novicida (Fn). Cells were fixed at 2 h after stimulation (nigericin) or 24 h post-Fn infection and analyzed for ASC specks by time-of-flight inflammasome evaluation (TOFIE). (For IL-1β release) HEK293T cells were transfected with 8 ng of myc-ASC with 100 ng NLRP3 or pcDNA3, 40 ng CASP1, and 200 ng IL1B. Eighteen hours post-transfection, cells were either stimulated with indicated agonists or left untreated. (A) Normalized ASC speck frequency (TOFIE) in comparison with normalized IL-1β release in reconstituted 293T cells following Fn U112 infection. (B) Normalized ASC speck frequency (TOFIE) in comparison with normalized IL-1β release in reconstituted 293T cells following nigericin stimulation. (C) Comparison of the ratio of normalized ASC specks frequency (TOFIE) with normalized IL-1β release following NLRP3 activation by nigericin stimulation and Fn U112 infection.