Fig. 2.

Caspase 1 is act ivat ed following LLOME and part icl e-induced lysosomal rupture in macrophages. a Caspase 1 act ivity in lysat es from LPS-pr imed BMDMs, tr eated with LLOME (0.5 mM) for 0.5 h to 6 h. Act ivity was measured using r eporter substrate Z-YVAD-AMC (60 μM), which upon hydrolysis by caspase 1produces a det ectabl e shift in RFU, n = 4 +/− standard error. b Confocal microscopy visual izing act ive caspase 1 in per itoneal macrophages i) l eft untreated or ii) treated with LLOME (0.5 mM) for 0.5 h or iii) 2 h. Cell s wer e incubat ed with the affinity-based FLICA probe, FAM-FLICA-FMK for 1 h, pr ior to washing and fixat ion. Images repr esentat ive of 3 independent exper iment s, scal e bar = 40 μm. c ELISA analysis of IL-1β released over t ime from LPS-pr imed BMDMs following treatment with LLOME (0.5 mM) for 0.5 h to 6 h, n = 4 +/standard error. d Confocal microscopy visual izing act ive caspase 1 in macrophages i) l eft untreated or treated for 2 h with ii) 500 μg/mL alum, iii)250 μg/mL sil ica , iv) 500 μg/mL PSNP. e Confocal microscopy visual izing act ive caspase 1 in macrophages i) treated with LLOME (0.5 mM) for 2 h or ii) pr e-treated with bafilomycin (250 nM) or iii) E64d (50 μM) pr ior to treatment with LLOME (0.5 mM) for 2 h. f ELISA analysis of IL-1β r el ease from LPSpr imed per itoneal macrophages pr e-treated with bafilomycin (250 nM) or E64d (50 μM) pr ior to treatment with LLOME (0.5 mM) for 6 h, n = 3 +/− standard error