Erythroid differentiation is dependent on arginine-derived polyamine biosynthesis but not transport. (A) Schematic representation of polyamine biosynthesis from arginine with key enzymes indicated in red (ornithine carboxylase [ODC], spermidine synthase [SRM], spermine synthase [SMS], spermidine/spermine-N1-acetyl transferase [SAT1]). Steps that are inhibited by α-difluoromethylornithine (DMFO), trans-4-methyl cyclohexylamine (MCHA), and N-(3-amino-propyl)cyclohexylamine (APCHA) are presented. N1, N11-diethylnorspermine (DENS), an activator of polyamine catabolism, is also indicated. (B) The impact of DFMO (1 mM) on rEPO-induced differentiation of CD34⁺ progenitors was monitored by evaluating CD11b/GlyA profiles at days 3 and 7. Representative dot plots (top) and quantification of relative levels at days 3 (n = 14 for GlyA, n = 12 for CD11b) and 7 (n = 11 for GlyA, n = 9 for CD11b) of differentiation are shown (bottom). (C) The impact of MCHA (100 μM) was evaluated as a function of CD11b/GlyA profiles (top) at day 3 and quantifications are shown (bottom, n = 7). (D) The impact of DENS (10 μM) on EPO-induced differentiation was evaluated at day 3 and representative plots (top) and quantifications (n = 7 for GlyA and n = 5 for CD11b) are shown (bottom). (E) APCHA (100 μM) was added to rEPO-induced progenitors and representative profiles (top) and quantifications (bottom, n = 7) are shown. (F) DFMO-treated progenitors were differentiated with rEPO in the presence or absence of putrescine (Put; 100 μM) or spermidine (Spd; 100 μM), and GlyA was evaluated at day 7 (top, green histograms). Quantification from 7 independent experiments is presented (bottom). (G) Erythroid differentiation was induced in the presence or absence of DFMO, spermidine, and AMXT-1501 (2.5 μM), an inhibitor of polyamine transport. Quantification of GlyA expression relative to control conditions is presented (bottom, n = 3). ∗P < .05; ∗∗P < .001; ∗∗∗P < .001; ∗∗∗∗P < .0001.