Caspase 8 is inactivated in Gpx4-deficient cells. (A) Immunoprecipitation of caspase 8 (IP:casp8) and immunoblot analysis of RIP1 and FADD in in vitro erythroid cultures treated with 4-OHT for 36 hours. Classical activation of necroptosis using zVAD/TAKi/TNF-α treatment in wild-type in vitro–cultured erythroid cells shows a strong interaction of caspase 8 with RIP1 and FADD upon 2 hours of stimulation. (B) Cleavage of caspase 8 (casp8) is blocked in in vitro–cultured Gpx4^Δ erythroid cells 36 hours after 4-OHT treatment, determined by immunoblot of caspase 8 when stimulated 2 hours with TNF-α in the presence of the TAK1 inhibitor 5Z-7- oxozeaenol (TAKi), whereas zVAD treatment completely inhibits caspase 8 cleavage. Concentration of GSSG (C) and the GSH/GSSG ratio (D) in peripheral blood cells of Gpx4^Δ mice and control littermates. (E) Detection of glutathionylated caspase 8 in peripheral CD71⁺/TER119⁺ cells from Gpx4^Δ mice. Cells were loaded with BioGEE, and immunoblot analysis was performed after immunoprecipitation with streptavidin (PD:strep). Data shown is representative of 4 independently analyzed mice of each genotype. DTT supplementation rescues cell death in erythroid cells (F) and restores caspase 8 cleavage upon TNF-α stimulation (G). DTT treatment does not inhibit lipid peroxidation (H) or ROS accumulation (I) in cultured erythroid cells of either genotype. (J) Our model proposes that in the absence of Gpx4, lipid peroxides and ROS can act as signaling molecules upstream of the necrosome independently of TNFR/FAS signaling. Loss of Gpx4 in the erythroid lineage leads to inactivation of caspase 8 via glutathionylation. ROS and lipid peroxides activate the RIP1/RIP3–containing necrosome and trigger necroptotic cell death. The presence of vitamin E (VitE) can compensate for Gpx4 deficiency.