Hypusination-induced OXPHOS is required for the erythroid commitment of hematopoietic progenitors. (A) Mitochondrial complexes (CI to CV) were monitored on progenitors treated with GC7 or after transduction with shCTRL or shDHPS vectors (day 3) using the OXPHOS monoclonal antibody cocktail (left). Quantification relative to control conditions was determined (n = 4 for GC7 and n = 3 for shRNA-transduced progenitors; middle and right, respectively). (B) Oxygen consumption rate (OCR), a measure of OXPHOS, was monitored on day 1 of erythroid differentiation in the absence or presence of GC7 (5 μM) on a Seahorse XFe96 analyzer after sequential injection of oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and Rotenone/Antimycin A (Rot/AntA; arrows, left). Mean basal OCR and SRC levels ± standard error of the mean (SEM) are presented (middle, n = 4). Representative energy plots of basal OCR and extracellular acidification rate (ECAR), a measure of glycolysis, are presented (right). (C) OCR was monitored on fluorescence-activated cell sorter–sorted shCTRL- and shDHPS-transduced progenitors at day 1 of differentiation (left). Basal OCR and SRC levels ± SEM were evaluated in 4 independent experiments (middle) and a representative OCR/ECAR energy plot is presented (right). (D) OCR was monitored on CD34⁺ progenitors differentiated with EPO for 24 hours in the absence or presence of GC7 (5 μM) and succinate (SUC, 5 mM) and representative graphs are shown (left). Basal OCR and SRC levels in 6 independent experiments are presented (right). (E) Erythroid differentiation in progenitors treated with EPO in the absence or presence of GC7 or succinate was evaluated at day 7 as a function of GlyA expression and representative histograms are shown (left). Quantification of the percentages of GlyA⁺ cells relative to control conditions are presented (right, n = 9). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, ∗∗∗∗P < .0001.