DHPS is required for erythroid commitment and differentiation. (A) Schematic representation of the 2-step process resulting in eIF5A^H. In the first step, DHPS catalyzes the addition of an aminobutyl moiety from the spermidine to the lysine 50 on eIF5A, generating an eIF5A intermediate. Subsequently, DOHH catalyzes the hydroxylation of the spermidine modification, generating an active hypusinated eIF5A. GC7 can inhibit the first step of this reaction. (B) The effect of GC7 on early erythropoiesis was evaluated by monitoring CD34/CD36 profiles of EPO-stimulated CD34⁺ progenitors in the absence (−) or presence (+) of GC7 (5 μM). Representative dot plots of IL3R⁻GlyA⁻ cells are shown and the percentages of IL3R⁻GlyA⁻CD34⁻CD36⁺ (CFU-E) are indicated (left). Quantification of CFU-E in 16 independent experiments are presented (right). (C) Representative dot plots of GlyA/CD11b profiles are presented at days 3 and 7 of EPO-induced differentiation in the absence or presence of GC7 (top). Quantification of GlyA⁺ and CD11b⁺ cells relative to levels in the absence of GC7 (indicated as “1”) are presented (bottom, n = 11-19). (D) CD34⁺ progenitors were differentiated in rEPO for 3 days and differentiation continued until day 7 in the absence or presence of GC7 (between days 3 and 7). Representative histograms showing GlyA expression at days 3 and 7 (with isotype controls, gray histograms) are presented and quantification of GlyA⁺ cells are presented relative to levels in the absence of GC7 (designated as “1,” bottom, n = 8). (E) CD34⁺ progenitors were transduced with shCTRL or shDHPS lentiviral vectors, harboring the enhanced GFP transgene and rEPO added 72 hours later. GFP expression was monitored at this time point (designated day 0), as well at days 3 and 7 of differentiation and representative are shown (top). Quantification of the evolution of GFP expression relative to day 0 (designated as “100%”) is presented for 5 donors (bottom). (F) shCTRL and shDHPS transduced progenitors were differentiated in the presence of rEPO for 3 days and representative CD34/CD36 profiles of IL3R⁻GlyA⁻ cells are presented (top). Quantification of IL3R⁻GlyA⁻CD34⁻CD36⁺ cells in shDHPS-transduced cells relative to shCTRL-transduced cells are shown (bottom, n = 9). (G) shCTRL and shDHPS transduced progenitors were differentiated for 3 days and representative GlyA/CD11 dot plots are shown (top). Quantification of GlyA⁺ and CD11b⁺ cells are presented relative to shCTRL conditions (bottom; n = 20 for GlyA, n = 21 for CD11b). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.