A Western Blot showing GSDME protein expression in L929sAhFas clones modified with CRISPR-Cas9 gene editing. B–D Flow cytometry analysis of the uptake of SB and 7-AAD in L929sAhFas WT and GSDME KO clones upon anti-Fas treatment. Results are presented as means ± SD (N = 3). B Representative flow cytometric plots with red arrows pointing to 7-AAD single positive cells. C Levels of 7-AAD (red) and SB (blue) positive cells given as percentage of total cell population upon anti-Fas treatment for two L929sAhFas GSDME WT clones. D Percentage of 7-AAD and SB positive cells given as percentage of total cell population upon anti-Fas treatment for two GSDME KO clones. E Western Blot showing doxycycline-dependent expression of GSDME in L929sAhFas iGSDME cells compared to GSDME expression in the L929sAhFas parental (P) cells and in L929sAhFas GSDME KOcl2. F–H Flow cytometry analysis of the SB and 7-AAD staining in L929sAhFas iGSDME with or without doxycycline pretreatment upon apoptosis induction by anti-Fas. Results are presented as means ± SD (N = 5). F Representative flow cytometry plots. G Percentage of 7-AAD and SB positive L929sAhFas iGSDME cells without doxycycline pretreatment upon anti-Fas treatment. H Effect of GSDME expression due to treatment with doxycycline on the amount of 7-AAD and and SB positive L929sAhFas iGSDME cells upon anti-Fas treatment. I–L Confocal images of L929sAhFas WT, GSDME KO cl1, iGSDME (− Dox) and iGSDME (+ Dox) cells upon anti-Fas treatment showing uptake of 7-AAD and SB over time. h hours, dox doxycycline, 7-AAD 7-aminoactinomycin D, SB SYTOX blue; scale bar = 100 µM. Videos available in supplementary material.