Lower oxidative phosphorylation, higher glycolysis, and ATP deficiency in PHOSPHO1-KO erythroblasts. (A) Erythroid progenitors from KO mice lost less lipid during the transition from the R2 to the R4 state than did cells from WT mice. Total lipid signals of R2 and R4 cells from lipidomic data shown in Figure 1B were used to calculate the ratios (n = 3 per group, mean + SEM). (B) WT and KO erythroblasts have similar sizes at the same differentiation stages. Representative forward scatter (FSC) and cell count of R2, R3, and R4 populations from WT and KO E14.5 fetal liver were plotted. Cells were gated as shown in Figure 1A (n = 3 per group). (C) AMP to ATP ratio is higher in KO 1-day–differentiated erythroblasts (n = 3 per group, mean + SEM). (D) Increased phospho-T172-AMPKα in 1-day in vitro–differentiated KO erythroblasts compared with WT. Ratio of phospho-AMPK to AMPK signal is indicated below, normalized to WT cells. Cells were isolated from E14.5 WT or KO fetal livers and expanded in maintenance medium for 1 day and cultured in differentiation medium for 1 day. (E) Basal and maximal OCR of 1-day–differentiated KO fetal liver erythroblasts is lower than that of WT erythroblasts; 1 μM oligomycin and FCCP, 0.5 μM rotenone and antimycin were used (n = 5 per group, mean ± SEM). (F) Higher basal glycolysis in KO erythroblasts. ECAR of 1-day in vitro–differentiated WT and KO erythroblasts was measured; 25 mM glucose, 1 μM oligomycin, and 50 mM 2-DG were sequentially added to the culture medium (n = 4 per group, mean ± SEM).