Figure 6.
Optogenetic activation of necroptotic effectors necroptosis. (A and B) Illustration of the necroptosis pathway and opto-hRIPK3 and opto-hMLKL constructs. (C) Quantification of necroptosis based on Annexin V acquisition in HEK293T cells expressing different opto-hRIPK3 versions or opto-hMLKL from 0 to 30 min after blue light illumination. (D and E) Time-lapse images of HEK293T cells undergoing light-induced necroptosis upon opto-RIPK3 (N-terminal, RHIM-deficient) or opto-hMLKL activation. Scale bar, 10 µm. (F) Dynamics of necroptosis induction via opto-hRIPK3 and opto-hMLKL activation. Scale bar, 10 μm. (G–I) Quantification of necroptosis in cells expressing WT, catalytically deficient or oligomerization-deficient opto-hRIPK3/opto-hMLKL or treated with 1 µM GSK’872 (RIPK3 inhibitor) or 5 µM necrosulfonamide (MLKL inhibitor) after 1 h of blue light illumination. (J) Confocal images of non-stimulated, early (pre-lytic) and late (permeabilized) necroptotic Caco-2 and HT-29 cells after activation of opto-hMLKL. (K) Quantification of Annexin V or CellTox staining in opto-hMLKL-expressing HT-29 cells after illumination with blue light. Scale bar, 20 µm. Data are representative (D, E, and J) or pooled from (C, F–I, and K) three independent experiments, each performed with triplicate technical replicates (n = 9), mean ± SD. ****, P < 0.0001 (two-tailed t test). TRAIL, TNF-Related Apoptosis Inducing Ligand; FADD, Fas Associated Via Death Domain; KD, kinase domain; PKD, pseudo kinase domain; NBB, N-terminal bundle and brace.

Optogenetic activation of necroptotic effectors necroptosis. (A and B) Illustration of the necroptosis pathway and opto-hRIPK3 and opto-hMLKL constructs. (C) Quantification of necroptosis based on Annexin V acquisition in HEK293T cells expressing different opto-hRIPK3 versions or opto-hMLKL from 0 to 30 min after blue light illumination. (D and E) Time-lapse images of HEK293T cells undergoing light-induced necroptosis upon opto-RIPK3 (N-terminal, RHIM-deficient) or opto-hMLKL activation. Scale bar, 10 µm. (F) Dynamics of necroptosis induction via opto-hRIPK3 and opto-hMLKL activation. Scale bar, 10 μm. (G–I) Quantification of necroptosis in cells expressing WT, catalytically deficient or oligomerization-deficient opto-hRIPK3/opto-hMLKL or treated with 1 µM GSK’872 (RIPK3 inhibitor) or 5 µM necrosulfonamide (MLKL inhibitor) after 1 h of blue light illumination. (J) Confocal images of non-stimulated, early (pre-lytic) and late (permeabilized) necroptotic Caco-2 and HT-29 cells after activation of opto-hMLKL. (K) Quantification of Annexin V or CellTox staining in opto-hMLKL-expressing HT-29 cells after illumination with blue light. Scale bar, 20 µm. Data are representative (D, E, and J) or pooled from (C, F–I, and K) three independent experiments, each performed with triplicate technical replicates (n = 9), mean ± SD. ****, P < 0.0001 (two-tailed t test). TRAIL, TNF-Related Apoptosis Inducing Ligand; FADD, Fas Associated Via Death Domain; KD, kinase domain; PKD, pseudo kinase domain; NBB, N-terminal bundle and brace.

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