Effects of GPX4 inhibition on human in vitro erythropoiesis from CD34⁺cells to orthochromatic erythroblasts. (A) Effect of GPX4 inhibitor, RSL3 at 1 µM, vs DMSO on cell counts from day 1 to day 18 during in vitro erythroid differentiation from peripheral blood CD34⁺ cells. Dead cells were excluded using trypan blue staining (n = 3). (B) Effect of GPX4 inhibitor, RSL3 at 1 µM, vs DMSO on clonogenic activity of erythroid progenitors. No differences in BFU-E and CFU-E counts were noticed (n = 3). (C) FCM dot plots assessing erythroid differentiation at early day 5 (CD34/CD36), intermediate day 7 and day 13 (CD71/GPA), and late day 15 (CD49d/Band 3) stages, in DMSO and 1 µM RSL3 conditions. Dot plots were obtained from 1 representative experiment (n = 3). (D-F) Comparative assessment of 3 features of orthochromatic erythroblast maturation between 1 µM RSL3 and DMSO-treated cells: mean fluorescence intensity (MFI) of CD49d assessed by FCM showing its kinetics of decrease between day 15 and day 18 (n = 3) (D), IFC analysis of cell size (E) and nucleocytoplasmic ratio (F), at day 18 to day 20 (depending on the kinetics of differentiation) (n = 5). None of these parameters were statistically different between DMSO and RSL3 conditions. A trend toward a higher cell size after RSL3 exposure was observed, but below significance threshold (P = .09). Statistical significance determined by Student t test. Error bars are SEM. NS, nonsignificant.