Fig. 1.

Impact of GSDME expression on apoptosis-driven secondary necrosis in L929sAhFas cells. a Expression and proteolytic cleavage of GSDME in L929sAhFas upon anti-Fas treatment. b Expression of GSDME in different L929sAhFas clones upon CRISPR/Cas 9 gene editing. c Cell death kinetics of parental, Gsdme WT and KO L929sAhFas clones measured by SB staining via flow cytometry. d Expression of GSDME in L929sAhFas iGSDME cells upon doxycycline treatment. Subsequent treatment with agonistic anti-Fas antibodies promotes the generation of the active 35 kDa N-terminal fragment (N-GSDME). e–h Flow cytometry analysis of L929sAhFas iGSDME cells with (L929sAhFas iGSDME+) or without (L929sAhFas iGSDME−) doxycycline-induced GSDME expression during apoptosis-driven secondary necrosis. e Representative flow cytometry dot plots after 4 h or 8 h treatment with anti-Fas. f Levels of secondary necrotic (SB+) cells, g cells exposing PS (AnnV+) and h PS single-positive (AnnV+ /SB−) cells in L929sAhFas iGSDME cells treated with anti-Fas. AnnV Annexin V; Dox doxycycline; GSDME, gasdermin E; KO knockout; LsFas L929sAhFas; NTC non-treatment control; Par parental; SB SYTOX Blue; WT wild-type