Figure S4.
Activation of zebrafish caspases by optogenetics and differential responses of apoptotic and necrotic cell death in epithelia. (A) Domain organization of zebrafish inflammatory (caspa and caspb) or apoptotic (caspase-8) caspases. (B) Representative images of hGSDMDtg HEK293T cells expressing opto-zfCaspa, opto-zfCaspb or opto-zfCaspase-8 before and after 30 min of blue light illumination (5 mW/cm2). CellTox staining (green) indicates membrane permeabilization. (C and D) Percentage of pyroptotic (C) or apoptotic (D) opto-zfCaspa or opto-zfCaspb-expressing (C) or opto-zfCaspase-8-expressing (D) hGSDMDtg HEK293T cells before and after blue light illumination (5 mW/cm2). Data is pooled from three independent experiments, mean ± SD. (E) Representative time-lapse images of a keratinocyte after activation of opto-zfCaspb. (F) Representative time-lapse images (showing DIC and mCherry channels) of Caco-2 cells undergoing light-induced pyroptosis, apoptosis and necroptosis in co-cultures with wild-type cells (related to Fig. 8, A and B). (G) Representative time-lapse images showing CellMask (purple), Annexin-V (blue) and CellTox (green) staining in pyroptotic (opto-hCaspase-1) and necroptotic (opto-hMLKL) cells being extruded from monolayer of WT cells. Left, images of the cells at the medial plane (5 µm above basal surface), right, apical plane (15 µm above basal surface, showing extruded cells). For opto-hMLKL cells, imaging was performed for 120 min to show CellTox influx during lytic necroptotic stage. (H) Time-lapse images of apoptotic cell (induced by opto-hCaspase-8 activation) in co-culture. Imaging was performed every 3 min for 12 h in total. Arrows indicate persisting apoptotic bodies in neighbors. (I and J) Percentage of extruded apoptotic cells during 12 h post apoptosis induction. (I) Cells were classified as “extruded,” if more than 50% of cell body or apoptotic fragments were expulsed from the monolayer. (J) Full and partial extrusion were defined as in Fig. 8. Data are pooled from two (I and J) independent experiments (n as indicated). All images are representative of at least three independent experiments (n = 3). Scale bars, 20 µm.

Activation of zebrafish caspases by optogenetics and differential responses of apoptotic and necrotic cell death in epithelia. (A) Domain organization of zebrafish inflammatory (caspa and caspb) or apoptotic (caspase-8) caspases. (B) Representative images of hGSDMDtg HEK293T cells expressing opto-zfCaspa, opto-zfCaspb or opto-zfCaspase-8 before and after 30 min of blue light illumination (5 mW/cm2). CellTox staining (green) indicates membrane permeabilization. (C and D) Percentage of pyroptotic (C) or apoptotic (D) opto-zfCaspa or opto-zfCaspb-expressing (C) or opto-zfCaspase-8-expressing (D) hGSDMDtg HEK293T cells before and after blue light illumination (5 mW/cm2). Data is pooled from three independent experiments, mean ± SD. (E) Representative time-lapse images of a keratinocyte after activation of opto-zfCaspb. (F) Representative time-lapse images (showing DIC and mCherry channels) of Caco-2 cells undergoing light-induced pyroptosis, apoptosis and necroptosis in co-cultures with wild-type cells (related to Fig. 8, A and B). (G) Representative time-lapse images showing CellMask (purple), Annexin-V (blue) and CellTox (green) staining in pyroptotic (opto-hCaspase-1) and necroptotic (opto-hMLKL) cells being extruded from monolayer of WT cells. Left, images of the cells at the medial plane (5 µm above basal surface), right, apical plane (15 µm above basal surface, showing extruded cells). For opto-hMLKL cells, imaging was performed for 120 min to show CellTox influx during lytic necroptotic stage. (H) Time-lapse images of apoptotic cell (induced by opto-hCaspase-8 activation) in co-culture. Imaging was performed every 3 min for 12 h in total. Arrows indicate persisting apoptotic bodies in neighbors. (I and J) Percentage of extruded apoptotic cells during 12 h post apoptosis induction. (I) Cells were classified as “extruded,” if more than 50% of cell body or apoptotic fragments were expulsed from the monolayer. (J) Full and partial extrusion were defined as in Fig. 8. Data are pooled from two (I and J) independent experiments (n as indicated). All images are representative of at least three independent experiments (n = 3). Scale bars, 20 µm.

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