Enucleation defect induced by GPX4 inhibition was not related to ferroptosis in human primary erythroblasts. (A) FCM quantification of lipid ROS using ¹¹C-BODIPY^581/591 staining in primary erythroblasts treated with RSL3 at 2 concentrations (1 µM and 3 µM) during 12 hours. H₂O₂ was used as positive control inducing a shift >70% (not shown). RSL3 induced a dose-dependent, statistically significant but weak increase in the percentage of positive cells (n = 3). Percentage of ¹¹C-BODIPY^581/591+ cells. DMSO vs 1 µM RSL3: 1% ± 0.1% vs 4% ± 0.5%, P < .05; 1 µM RSL3 vs 3 µM RSL3: 4% ± 0.5% vs 8% ± 0.7%, P < .01. (B) Histogram showing quantification of enucleated GPA⁺/Hoechst⁻ cells (%) at day 20 in DMSO and 1 µM RSL3-treated cells, with and without ferroptosis inhibitors (100 µM α-tocopherol, 2 µM ferrostatin, 5 µM deferoxamine), 20 µM apoptosis inhibitor (QVD), 10 µM necroptosis inhibitor (Necrox), and 5 mM autophagy inhibitor (3MA). There was no statistically significant difference in the enucleation rate between DMSO and all of the different inhibitors tested (n = 3). (C) Induction of membrane lipid peroxidation assessed by FCM and ¹¹C-BODIPY^581/591 staining after inhibition of GPX4 using 1 µM RSL3 and inhibition of the GPX activity of PRDX6 using 40 µM MSA (n = 3). DMSO vs RSL3 and MSA, P < .05; RSL3 vs MSA, P: NS; RSL3 or MSA vs RSL3 + MSA, P < .05. *P < .05; **P < .01. Error bars are SEM.