GPX4 inhibition led to disruption of lipid rafts at cleavage furrow and to decrease in myosin phosphorylation that was partially restored by cholesterol. (A) Representative image of DAPI, AlexaFluor 488–conjugated pMRLC, AlexaFluor 647–conjugated CTB staining using fluorescence microscopy in DMSO and 1 µM RSL3-treated cells at day 18 (n = 3). (B) Histogram showing the significant decrease in CTB MFI measured by FCM at day 20 in 1 µM RSL3-treated erythroblasts in comparison with DMSO (n = 3). Mean MFI: 80 ± 5 (DMSO) vs 37 ± 12 (RSL3), P < .05. (C, left) Representative pMRLC immunoblot of erythroblasts treated with DMSO or 1 µM RSL3 during the enucleation stage at day 18. (C, right) Quantification of the decrease in pMRLC induced by 1 µM RSL3 exposure, using the pMRLC/MRLC vs GAPDH ratio (n = 3): 0.69 ± 0.05 vs 0.34 ± 0.06; P < .001. (D) Histogram showing MFI of pMRLC, measured by FCM in fixed and permeabilized erythroblasts in DMSO and 1 µM RSL3 conditions, during the enucleation stage (n = 3). P < .05. (E) Addition of 10 µM cholesterol at day 14 corrected the CTB decrease induced by RSL3 exposure, as assessed by FCM (n = 3). Day 20 erythroblasts, CTB MFI RSL3 vs RSL3 + CHOL: 3.9 ± 0.3 vs 5.9 ± 0.2, P < .05. (F, left) Representative pMRLC and GPX4 immunoblot of erythroblasts treated with DMSO or 1 µM RSL3, with and without 10 µM cholesterol, during the enucleation stage (n = 3). Catalase, MRLC, and GAPDH were used as load controls. (B, right) Densitometric pMRLC/MRLC vs GAPDH ratio between RSL3 and RSL3 + CHOL from 3 independent experiments: 0.137 ± 0.05 vs 0.507 ± 0.06, P < .001. *P < .05; **P < .01. Error bars are SEM.