VHH_ASC-EGFP assembles along ASC filaments in response to inflammasome triggers. (A–D) THP-1 cell lines inducibly expressing VHH_ASC-EGFP or VHH NP1-EGFP were differentiated and VHH expression induced for 24 h. Cells were left untreated (A), treated with LPS for 3 h and nigericin for 45 min to trigger NLRP3 inflammasomes (B), treated with LPS for 3 h and transfected with DNA for 4 h to trigger AIM2 inflammasomes (C), or treated with PA and LFn-MxiH WT in the presence of 30 µM z-YVAD-cmk for 2 h to trigger NAIP/NLRC4 inflammasomes (D). Cells were fixed and stained for DNA and ASC; images of single planes were recorded by spinning disc confocal microscopy. Bars, 10 µm. Representative images from at least three experiments are shown. (E–G) THP-1 cell lines were differentiated, VHH expression induced, and NLRP3, AIM2, and NLRC4 inflammasomes activated or cells left untreated as in A–D, but in the presence of 50 µM z-YVAD-cmk. Cells were fixed and mounted with mounting medium containing DAPI; Z stacks were recorded by spinning disc confocal microscopy. At least 300 cells per condition were analyzed and cells containing green-fluorescent filaments or foci were manually counted. One typical experiment representative of at least three independent experiments was quantified.