Fig. 3: A cell-based screen identifies phenytoin as an inhibitor of necroptosis.

a, b HT29 cells were treated with TNFα/smac mimetic/zVAD-fmk to induce necrosulfonamide (NSA)-sensitive necroptosis over 4, 16 and 24 h. Cell cultures were split for FACS analysis (a) and western blotting (b). Note that cells that exhibit pMLKL-positivity in western blots (16 h with NSA) did neither lose plasma membrane integrity nor became positive for annexin V between 16 and 24 h. c Hypothesis-based screen for clinically available inhibitors of plasma membrane channels based on 7-AAD/annexinV positivity in L929 cells 48 h following TZ-treatment. Nec-1, Nec-1s, and ponatinib served as positive controls. d Verification of phenytoin in HT29 cells as an inhibitor of TSZ-induced necroptosis in direct comparison with Nec-1s. e Phenytoin prolongs survival after TNFα-induced SIRS. e−h Phenytoin significantly protects from renal ischemia-reperfusion injury. Statistical significance was indicated: n.s.: not statistically significant, p < 0.05 (*), p < 0.01 (**), n = 8 mice per group unless otherwise indicated. Scale bars = 50 µm