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

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© 2022 Shkarina et al.

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Figure S1. — Activation of optogenetic inflammatory caspases in human cells. (A) Western blot analysis of the expression of inflammatory (hCaspase-1, hCaspase-4, and hCaspase-5) and apoptotic (hCaspase-8 and hCaspase-9) caspases and hGSDMD in the cell lines used in the study. (B) To determine the phototoxicity threshold, HEK293T cells expressing mCherry-Cry2olig were illuminated with blue lights of different power density every 15 s for 1 h, and percentage of cells with altered morphology was quantified before and after illumination. (C) Representative images of cells from A before and after 1 h after illumination with blue light (5 mW/cm²). Insets show the morphology of transfected (mCherry⁺) and non-transfected (mCherry⁻) cells. (C) Representative images of HEK293T cells expressing different opto-hCaspase-1, -4, and -5 versions before and after illumination (488 nm, 5 mW/cm²). (D) Normalized fluorescence intensity of the mCherry, Annexin-V, and CellTox signals in cells expressing CARD-deficient opto-hCaspase-1 and undergoing light-induced pyroptosis. Mean ±SEM, n = 5 cells. (E) Representative images of wild-type (i.e., naturally GSDMD-deficient) HEK293T cells expressing opto-hCaspase-1, -4 or -5 (red) before and after illumination (488 nm, 5 mW/cm²). (F) Quantification of apoptotic, pyroptotic, or viable cells before and after illumination among wild-type or hGSDMD^tg HEK293T cells expressing opto-hCaspase-1, -4, or -5. Images show morphological features and CellTox staining of the three categories of cells (viable, apoptotic, and pyroptotic). (G) Representative images of wild-type HEK293T cells expressing opto-hCaspase-1/-4/-5 and the genetically encoded caspase-3/7 activity reporter VC3AI (green) before and after illumination (488 nm, 5 mW/cm²). (H) Representative images of different human epithelial cell lines expressing opto-hCaspase-1/-4/-5 before and after 1 h of illumination (488 nm, 5 mW/cm²). Scale bars, 20 µm. All data are representative of at least two (A) or three (C–H) independent experiments, each done with triplicate technical replicates (n = 6 and n = 9).