(A) Wild type macrophages stably expressing ASC-CFP were treated with L. monocytogenes expressing GFP, transfected with poly(dA:dT) or left untreated. 6 hours after stimulation, the formation of ASC complexes was quantified using confocal microscopy. (B) ASC-CFP expressing macrophages were treated with ATP/LPS, decreasing amounts of L. monocytogenes (MOI 20 – 0.625 in steps of 50%), a mutant L. monocytogenes strain lacking LLO (Listeria ΔLLO, MOI 20) or transfected with poly(dA:dT) and formation of ASC complexes was quantified using epifluorescence microscopy. (C) Wild type macrophages or ASC-deficient macrophages were treated with the stimuli as above (L. monocytogenes MOI of 10). 6 hours after stimulation pro-caspase-1 (pro-casp1) expression was assessed in cell lysates (cells), while caspase-1 activation (casp1 p10) was assessed in the supernatant (SN) using western blot. (D) Wild type macrophages were treated with 20 ng /ml cycloheximide or left untreated. After 30 min cells were stimulated as indicated (L. monocytogenes and S. enterica MOI of 10) and caspase-1 expression and activation was assessed as above. (E) Wild type, (F) NLRP3- or (G) IPAF-deficient macrophages were stimulated as indicated (L. monocytogenes / S. enterica MOI of 50, 10 or 1) and 6 hours after stimulation caspase-1 expression and activation was assessed. One representative experiment out of three (A, B, D) or four (C, E, F, G) independent experiments is depicted.

(A) Wild type macrophages were transduced with two individual shRNA targeting AIM2 or two control shRNA. 7 days after transduction, β-Actin and AIM2 expression was analyzed by western blot. (B) Wild type macrophages or NLRP3-deficient macrophages were transduced using shRNA targeting AIM2 or a control shRNA and 7 days after selection cells were stimulated as indicated (L. monocytogenes / S. enterica MOI of 50, 10 or 1). 6 hours after stimulation caspase-1 expression and activation was assessed by western blot. (C) NLRP3-deficient macrophages transduced as in (B) were stimulated with L. monocytogenes as indicated and 24h after transfection viable cells were labeled using calcein AM and subsequently visualized using fluorescent microscopy. One representative visual field (20 × objective) for the conditions tested is depicted in the left panel. In addition, cells in two independent visual fields (20 × objective) were counted using ImageJ. Data are depicted as mean + SD. (D) Lysates of macrophages as in (C) were prepared and colony forming units of L. monocytogenes were quantified. Data from two independent replicates are depicted as mean + SD. One representative experiment out of three independent experiments is depicted (A–D).