Fig. 4: FLAG-MLKL-induced cell death induces signaling to bystander cells.

A Representative images of HEK293T cells were separately transfected with FLAG-MLKL-CRY2olig-mCh or GCaMP6, then mixed at a ratio of 1:5, respectively. Minutes after light stimulation, FLAG-MLKL-expressing cells show membrane rupture (white arrow). Within 6 s after rupture, the surrounding GCaMP6-expressing bystander cells show an increase in Ca²⁺. B Representative images of HEK293T cells separately transfected with FLAG-MLKL-CRY2olig-mCh or PKCγ-C2-GFP, then mixed at a ratio of 1:5. Minutes after light onset, FLAG-MLKL-induced cell death (rounded cell on right side of left panel) triggers transient activation and membrane recruitment of PKC in bystander cells. The zoom of the area in a white rectangle is shown on the right. C Quantification of change in GCaMP signal (∆F/F₀) in bystander cells in proximity to dying Flag-MLKL-expressing cell as in (A). Time 0 marks the beginning of rise in GCaMP signal, as defined by an increase in baseline > 2 standard deviations from the mean. Thirteen cells were quantified from n = 3 independent experiments. D Quantification of the half-time of recovery from peak GCaMP signal from responding cells in (C). E Quantification of change in cytosolic intensity of PKCγ-C2-GFP in bystander cells in proximity to dying FLAG-MLKL-expressing cells as in (B). Shown are eight cells from n = 3 independent experiments. F Quantification of the half-time of recovery from peak cytosolic depletion of PKCγ-C2-GFP, for cells shown in (E). G, H Apyrase addition blocks Ca²⁺ and PKCγ responses in unattached bystander cells. The experiment was performed as in (A), except cells were incubated with or without 10 U/ml apyrases and used a far-red MLKL (FLAG-MLKL-CRY2olig-miRFP670_nano) to simultaneously assess MLKL membrane recruitment, nuclear PI staining, and either GCaMP6 or C2γ-GFP signal. Upon cell rupture (white arrowheads), nearby GCaMP-expressing observer cells (yellow arrowheads) show an increase in Ca²⁺ in the absence, but not presence, of apyrase. Cells that are attached to the dying cell (pink arrows) were not affected by apyrase treatment. Representative images are shown in (G), with quantification of the GCaMP (max ∆F/F₀) and PKCγ (max cytosolic decrease) responses in bystander cells within 150 µm in (H). Data were obtained from 3 independent experiments. Scale bar, 10 μm (all images).