Optogenetic activation of apoptotic initiator caspases. (A and B) Schematic representations of intrinsic and extrinsic apoptosis induction and full-length or truncated opto-hCaspase-8 and opto-hCaspase-9. (C) HEK293T cells expressing opto-hCaspase-8/9 versions were illumination with blue light and percentage of apoptotic cells over time determined based on morphology (cell shrinking, blebbing, nuclear fragmentation). (D) Time-lapse images and Annexin V acquisition of HEK293T cells expressing DED-deficient opto-hCaspase-8 and CARD-deficient opto-hCaspase-9 after illumination. Scale bar, 10 µm. (E) Images activation of caspase-3/7 reporter in opto-hCaspase-8/9-expressing HEK293T cells before and after 1 h of blue light illumination. Scale bar, 10 µm. (F) Quantification of apoptotic cells from D over time (in 1-min intervals). (G–I) Quantification of apoptosis induction in cells expressing WT (G and I) or mutated (G) opto-hCaspases or treated with Z-VAD (H) 1 h after blue light illumination. Data are representative of (D) or pooled from (C and E–I) three independent experiments, each performed with triplicate technical replicates (n = 9). Each data point corresponds to independent technical replicate. Mean ± SD; P < 0.0001 (two-tailed t test). TNFR, TNF-receptor; MOMP, mitochondrial outer membrane permeabilization; DED, death-effector domain.