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

Figure S2.

Figure S2.

Optogenetic induction of pyroptosis in human and mouse macrophage-like cells, and sublethal induction of pyroptosis. (A) Representative image showing heterogenous mCherry expression levels in U937 cells stably transduced with opto-hCaspase-1 after doxycycline (dox) treatment. Scale bar, 50 µm. (B) Percentage of mCherry-positive cells in differentiated dox-treated U937 cell lines expressing indicated constructs. Data is pooled from two independent experiments, four fields of view per experiment, mean ± SD. (C) Schematic presentation of mouse opto-mCaspase-1 and opto-mCaspase-11 constructs. Both constructs contain CARD-deficient mouse caspase-1 or 11 proteins N-terminally fused to mCherry-tagged Cry2olig via a GGGS linker. (D) Representative images of wild-type or GSDMD-transgenic HEK293T cells expressing opto-mCaspase-1 or opto-mCaspase-11 before or 30 min after illumination (488 nm, 5 mW/cm2 every 15 s). Cells were imaged in presence of CellTox (green) and Annexin-V (blue) to visualize membrane permeabilization and PS exposure. Scale bars, 10 µm. (E) Percentage of CellTox-positive hGSDMDtg HEK293T cells expressing wild-type or catalytically-deficient opto-mCaspase-1 or opto-mCaspase-11 before and after illumination (488 nm, 5 mW/cm2 every 15 s). Mean ± SD, pooled from three independent experiments. ****, P < 0.0001; ***, P < 0.001 (two-tailed t test). (F and G) Representative images of RAW 264.7 cells and primary murine bone marrow derived macrophages (BMDMs) expressing Cry2olig, opto-mCaspase-1 or opto-mCaspase-11 before and after illumination (488 nm, 5 mW/cm2) in presence of CellTox and Annexin V. Scale bars, 20 µm (F) and 10 µm (G). (H) Representative images of cells not affected by the first illumination pulse (viable, DRAQ7−) or undergoing complete pyroptosis (pyroptotic, DRAQ7high). Related to Fig. 3 E. Scale bar, 20 µm. (I) HaCaT cells expressing opto-hCaspase-1 were transiently illuminated with low-intensity 488 nm light (3 × 0.2 mW/cm2) at t = 3min and imaged for 6 h. DRAQ7 influx (turquoise) marks the cells with initial sub-lethal membrane permeabilization, which was then repaired. Arrows indicate dividing parent cell and its daughters. Scale bar, 20 µm. (D–G) Data are representative of or combined from three independent experiments, each done with triplicate technical replicates (n = 9). H and I are representative of six independent experiments (n = 6).