RIP3-dependent necroptosis, but not ferroptosis, causes cell death in Gpx4-deficient erythroid progenitor cells. Erythroid cells were differentiated in vitro from Gpx4^F/F and Rosa26-CreER^T2/Gpx4^F/F bone marrow, and deletion was induced by 4-OHT after 24 hours of culture in the presence of EPO. Microscopic images of in vitro–differentiated erythroid cells from Gpx4^F/F (A) and Rosa26-CreER^T2/Gpx4^F/F (Gpx4^Δ) (B) bone marrow 48 hours after the induction of deletion with tamoxifen. (C) Schematic illustration of programmed cell death pathways. Ferroptosis (left) is triggered by an iron-dependent accumulation of lethal ROS and lipid peroxides in cells, which can be inhibited via iron (Fe) chelators such as DFO. Ferroptosis can be induced by erastin, which inhibits cellular cysteine (Cys) uptake and thus limits the production of intracellular GSH, or by Ras synthetic lethality molecule 3 (RSL3) via inhibition of Gpx4, leading to increased lipid peroxidation and ROS accumulation. Fer-1 and Lip-1 inhibit ferroptosis via inhibiting the lipid peroxidation. Apoptosis and necroptosis (right) are mainly regulated via TNFR1 signaling. Upon TNF binding, TNFR1 undergoes a conformational change, activating 2 possible cell death execution mechanisms: caspase-dependent or caspase-independent. Normally, caspase 8 triggers apoptosis by activating the classical caspase cascade. It also cleaves, and hence inactivates, RIP1 and RIP3. If caspase 8 is inhibited (eg, via zVAD), phosphorylated RIP1 and RIP3 engage the effector mechanisms of necroptosis. (D) Percentage of viable in vitro–differentiated erythroid cells counted via trypan blue exclusion 48 hours after the induction of deletion in the presence of α-tocopherol (the most prominent member of the vitamin E family), the ferroptosis inhibitors Fer-1 and Lip-1, the iron chelator DFO, the pan-caspase inhibitor zVAD, the RIP1 kinase inhibitor nec-1, or recombinant soluble TNFR2 (etanercept). Data are mean ± SE; n ≥ 6; *P < .05 by Student t test. (E) Absence of Gpx4 and RIP3 is verified by immunoblot analysis. (F) Percentage of viable in vitro–differentiated erythroid cells from Gpx4^F/F, Rip3^−/− (Gpx4^F/FRip3^Δ), Rosa26-CreER^T2/Gpx4^F/F (Gpx4^Δ), and Rip3^−/−/Rosa26-CreER^T2/Gpx4^F/F (Gpx4^ΔRip3^Δ) 48 hours after the induction of deletion. Data are mean ± SE; n ≥ 8; ***P < .001 by ANOVA/Bonferroni. (G) Flow cytometry analysis of in vitro–cultured erythroid cells 36 hours after the 4-OHT treatment to analyze necrotic cells (AnnexinV⁻PI⁺). Data are mean ± SE; n ≥ 4; *P < .05 by ANOVA/Bonferroni. (H) Deletion of Rip3 significantly improved the formation of erythroid o-dianisidine–positive Gpx4^Δ colonies, similar to α-Toc supplementation. Data are mean ± SE; n ≥ 3; *P < .05, **P < .01, ***P < .001 by ANOVA/Bonferroni. ANOVA, analysis of variance.