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. 2022 Apr 14;221(6):e202109038. doi: 10.1083/jcb.202109038

Figure 1.

Figure 1.

Development and validation of optogenetically activated human inflammatory caspases. (A) Inflammasome pathway, Cry2-mediated clustering of inflammatory caspases. (B and C) Domain architecture of human inflammatory caspases and opto-hCaspase constructs. (D) Quantification of CellTox-positive (permeabilized) human GSDMD-transgenic (hGSDMDtg) HEK293T cells expressing opto-hCaspases untreated or illuminated with blue light every 30 s. Here and after, only cells expressing detectable levels (mCherry+) of optoCDEs at t = 0 were quantified, and unless stated otherwise 488 nm light at 5 mW/cm2 was used for all assays. (E) Time-lapse images of hGSDMDtg HEK293T cells after blue-light activation of Cry2olig or opto-hCaspase-1, -4, or -5 showing PS exposure (Annexin V) and membrane permeabilization (CellTox Green). Scale bar, 20 µm. (F) DRAQ7 uptake over time (quantified in 1-min intervals) in hGSDMDtg HEK293T cells expressing indicated constructs. (G and H) Percentage of CellTox+ hGSDMDtg HEK23T cells expressing WT or mutant opto-hCaspases before and after illumination; or after mock (NT) or Z-VAD-fmk treatment. (I) Percentage of CellTox+ cells in cell lines expressing indicated constructs at 1 h post illumination. All data are shown as mean ± SD (D and G–I) or mean ± SEM (F) and are representative of three independent experiments (E) or pooled from three independent experiments (D, F, G, H, and I), each with three technical replicates per experiment (n = 9 in total). Each dot represents an independently stimulated field of view. ****, P < 0.0001; **, P < 0.01 (two-tailed t test). PAMPs, pathogen-associated molecular patterns; LPS, lipopolysaccharide; DAMPs, danger-associated molecular patterns; CARD, Caspase recruitment domain; p20, catalytic p20 domain; p10, catalytic p10 domain.