Figure S3.

Optogenetic activation of apoptosis, necroptosis and pyroptosis in individual cells in 2D and 3D. (A) Normalized nuclear DRAQ7 intensity of eight unstimulated directly adjacent neighbors of the cells selectively stimulated with blue light. Related to Fig. 4 A. Grey lines indicate individual cell traces. Vertical blue lines indicate the timing of each of the three local blue light stimulations. (B) Representative time-lapse images of partial cell ablation in non-lumenized Caco-2 spheroid. Blue sphere indicates the region where the stimulation was performed at t = 5 min. Scale bar, 50 µm. Representative of three independent experiments. (C) Representative images of HEK293T cells expressing full-length or CARD/DED-deficient opto-hCaspase-8 or opto-hCaspase-9 before and after illumination (488 nm, 5 mW/cm² every 15 s). Arrows indicate apoptotic blebbing. Scale bars, 20 µm. (D) Quantification of cells from C expressing opto-hCaspase-8 or opto-hCaspase-9 and displaying apoptotic blebbing alone, or blebbing and Annexin V staining. Cells were stimulated with blue light every 3 min, and images were acquired for 3 h. (E) U937 or HaCaT cells expressing opto-hCaspase-8, or Caco-2 cells expressing opto-hCaspase-9 were treated with the SMAC mimetic AZD5582 and stimulated with blue light for 1 h, and percentage of cells displaying apoptotic morphology was quantified before and after illumination. Mean ± SD. Data in C–E are pooled from three independent experiments. ***, P < 0.001 (two-tailed t test). (F) Representative images of HEK293T cells expressing wild-type or RHIM-deficient opto-hRIPK3 (C-terminally fused to Cry2olig) in absence of blue light illumination (left). Percentage of cells with mCherry clusters in HEK293T cells expressing different opto-hRIPK3 versions (right). Scale bars, 10 μm. (G) Representative images of cells expressing wild-type C-terminal opto-hRIPK3 and displaying apoptotic morphology in absence of blue light (left). Percentage of spontaneously apoptotic cells in HEK293T cells transfected with different opto-hRIPK3 versions (right). Scale bars, 10 µm. (H) Representative images of HEK293T cells expressing different opto-hRIPK3 versions (co-transfected with human hMLKL) or opto-hMLKL before and after stimulation with blue light (5 mW/cm²). Cells were imaged in presence of Annexin V (blue) and CellTox (green) to visualize early (pre-lytic) and late (lytic) necroptosis. Scale bars, 20 µm. (I) Percentage of Annexin-V-positive HEK293T cells expressing opto-hRIPK3 versions before and after illumination, in absence of hMLKL co-expression. Data in F–I are pooled from two (E) or three (B–H) independent experiments, performed with triplicate technical replicates (D, E, and I) or quadruplicate technical replicates (F–H). n = 6 (E), n = 9 (D and I), and n = 12 (F–H). Mean ± SD (two-tailed t test). Scale bars, 10 µm (A and B) or 20 µm (C).