DHPS-mediated hypusination of eIF5A regulates protein synthesis in early erythroid progenitors. (A) Hypusination was evaluated in progenitors differentiated in the presence or absence of EPO (day 4) and representative immunoblots of eIF5A^H, eIF5A, and actin are shown (left). Quantification of eIF5A/actin (middle) and eIF5A^H/eIF5A (right) ratios are presented relative to levels in the presence of EPO (n = 7). (B) Proerythroblasts (Pro), basophilic (Baso), polychromatic (Poly), and orthochromatic (Ortho) erythroblasts were sorted based on their GLUT1/CD49d prolife at day 7 of differentiation and hypusination was monitored by immunoblotting (left). eIF5A^H was quantified relative to total eIF5A levels (right, n = 5). (C) Protein synthesis was monitored at the indicated day of erythroid differentiation by O-propargyl-puromycin (OPP) labeling and representative histograms and MFI are indicated (left). Quantification of MFIs relative to day 4 are presented for 3 donors (right). (D) Protein synthesis was monitored by OPP labeling in erythroblast subsets 24 hours after sorting (as in panel B, left). Quantification of MFIs relative to proerythroblasts are presented for 3 donors (right). (E) Hypusination was evaluated after 3 days of EPO stimulation in the absence (−) or presence (+) of GC7 (5 μM) and representative immunoblots of eIF5A^H, eIF5A, and actin are shown (left). Quantification relative to levels in the absence of GC7 are presented (right, n = 9). (F) Hypusination in shCTRL- and shDHPS-transduced progenitorsevaluated at day 3 of differentiation after sorting based on GFP expression and representative immunoblots are shown (left). Quantification of the relative levels of eIF5A^H/eIF5A is presented (right, n = 5). (G) CD34⁺ progenitors were differentiated in the presence of EPO, together with GC7 (5 μM) or cycloheximide (CHX; 1 μM). Protein synthesis was evaluated at day 1 of differentiation and a representative histogram is shown (left). Quantification of protein synthesis relative to control conditions is presented (right, n = 8). (H) shCTRL- and shDHPS-transduced progenitors were sorted 72 hours after transduction based on GFP expression. Protein synthesis evaluated 24 hours after the addition of EPO and a representative histogram (left) as well as quantification (right, n = 3) are presented. (I) CD34⁺ progenitors were differentiated with EPO in the absence or presence of GC7 (5 μM) for 2 days and protein expression was evaluated by mass spectrometry–based quantitative proteomics. A volcano plot shows differences in protein expression (log2 fold change) induced by GC7, and the identity of specified downregulated and upregulated proteins are noted. Statistical significance of relative protein expression is computed via 2-sample moderated t test, and proteins with an false discovery rate (FDR) adjusted (adj.) P < .05 are colored in red. (J) Overrepresentation analyses of gene ontology for nonredundant biological processes were evaluated for significantly upregulated and downregulated and enrichment scores are indicated. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.