Enucleation impairment induced by GPX4 inhibition and gene silencing. (A, left) Representative Band 3/Hoechst dot plots obtained at day 20 in GPA⁺ control and 1 µM RSL3-treated cells using FCM (n = 5). (A, right) Curves showing quantification of enucleated GPA⁺/Hoechst⁻ cells (%) from day 16 to day 20 in 1 µM RSL3 and control treatments (n = 5). Percentage of enucleated cells. DMSO vs RSL3: day 18, 49% ± 4% vs 31% ± 3%, P < .01; day 20, 78% ± 5% vs 39% ± 5%, P < .01. (B, left) May-Grünwald-Giemsa (MGG) staining (magnification ×100) of DMSO or 1 µM RSL3-treated cells. Images shown were taken at day 18 and were representative of 3 independent experiments. (B, right) Cytological count after MGG staining of orthochromatic erythroblasts and reticulocytes (%) in 1 µM RSL3 and control conditions (n = 3). Percentage of RET. DMSO vs RSL3: 43% ± 6% vs 10% ± 3%, P < .01. (C, left) histogram representing the percentage of decrease in day 20 enucleation observed after transduction of 3 different shRNA against GPX4 (SH1, SH2, SH3) in comparison with control (ShSCR) (n = 3). Percentage of enucleation decrease in comparison with SCR. SH1: 52% ± 3%; SH2: 21% ± 2%; SH3: 49% ± 4%, SCR vs each SH: P < .01. (C, right) A representative histogram of Hoechst staining at day 20 in SCR and SH1-transduced cells. **P < .01. Error bars are SEM.