Fig. 2: FLAG-MLKL allows improved light control of cell death. | Cell Death Discovery

Fig. 2: FLAG-MLKL allows improved light control of cell death.

From: Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains

Fig. 2

A Comparison of expression of MLKL-CRY2olig-mCh (‘MLKL-CRY2olig’) and FLAG-MLKL-CRY2olig-mCh in HEK293T cells. Cells were transfected using Lipofectamine 2000, and mCherry fluorescence was quantified 24 h post-transfection. Control cells expressing CRY2olig-mCherry were also quantified for comparison. a.u., arbitrary units; n.s., p-value = 0.70; ***, p-value < 0.001, two-tailed unpaired t-test. B Representative images of cells expressing FLAG-MLKL before and after light exposure. HEK293T cells transfected with FLAG-MLKL-CRY2olig-mCh were incubated with 1 µg/ml PI and exposed to blue light (100 ms pulse, 488 nm, every 30 s). FLAG-MLKL translocates to the plasma membrane and results in cell rounding. PI staining can be observed as a sudden appearance of nuclear fluorescence, seen here at 19.5 min. C Quantification of loss of cytosolic FLAG-MLKL upon light-induced recruitment to the PM. HEK293T cells expressing FLAG-MLKL-CRY2olig-mCh were exposed to blue light 24 h after transfection. A large fraction of FLAG-MLKL translocates from the cytosol to the plasma membrane with a half-life of 2.6 s. Data represents mean ± s.e.m. of six cells from three independent experiments. D Correlation of expression level and cell fate. Graph shows mean total fluorescence of HEK293T cells expressing indicated constructs exposed to a single pulse of blue light and monitored for 30 min for PI-positive staining. (a.u., arbitrary units). n = 60–175 cells quantified per construct. Data are representative of n = 3 independent experiments. E Graph showing the relationship between expression level and time until cell death (PI staining), for cells treated as in (D) (35 cells quantified). Data are representative of n = 3 independent experiments. F Representative images (left) and quantification (right) of HEK293T cells expressing FLAG-MLKL-CRY2olig-mCh and GCaMP6. Cells show a large increase in GCaMP6 signal within 60 s after light exposure. Graph shows mean ± s.e.m., n = 6 cells. G HEK293T cells expressing indicated constructs and incubated with 1 µg/ml PI were quantified for cell death (PI-positive staining) in the absence of blue light stimulation using an IncuCyte imager. Graph shows mean ± s.e.m. of triplicate samples. Data are representative of n = 2 independent experiments. H Cells expressing indicated constructs incubated with PI and exposed to light (488 nm pulse every 15 min) were quantified for cell death (PI staining) using an IncuCyte imager. Data represent mean and s.e.m. of quadruplicate samples, and are representative of n = 3 independent experiments. I Representative confocal images of HEK293T cells expressing FLAG-MLKL exposed to blue light. Cells also contained 400 nM Sytox green and Cy5-Annexin V. J Focal application of blue light to a single cell expressing FLAG-MLKL-CRY2olig-mCh. Blue light was applied only to the area indicated by the blue box using an Andor Mosaic digital mirror device, while the entire region was imaged using the mCherry channel. Scale bar (all images), 10 μm.

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