PHOSPHO1 KO erythroblasts are deficient in choline and increase glycolysis to produce serine and/or glycine. (A) Left, Choline abundance is higher in WT 1-day–differentiated erythroblasts. Right, The phosphocholine to choline ratio is higher in R2, R3, and R4 groups of KO erythroblasts compared with WT cells. Metabolites were normalized to total lipid (n = 3 per group, mean + SEM). (B) Crosstalk of phosphocholine catabolism and glycolysis. Production of serine and glycine links the 2 pathways. Enzymes involved in the steps are labeled. (C) Increased mRNA expression of genes encoding proteins involved in glycolysis and serine/glycine production in 1-day–differentiated KO erythroblasts. Lineage-negative cells from WT and KO E14.5 fetal livers were isolated and cultured in maintenance medium for 1 day and differentiation medium for 1 day prior to RNA analysis; mRNA levels are normalized to that of 18s rRNA. (D) Phospho-T172-AMPKα signal is higher in 1-day–differentiated KO erythroblasts than in WT cells, and the signal is reduced in cells treated with 0.1 mM serine or 0.1 mM glycine. Relative phospho-AMPK to AMPK signal ratio is depicted below. (E) Proliferation is increased to normal in KO erythroblasts cultured in differentiation medium containing 0.2 mM serine (n = 3 per group, mean + SEM). (F) Proliferation is increased to normal in KO erythroblasts cultured in differentiation medium containing 0.1 mM glycine (n = 3 per group, mean + SEM). (G) Increased enucleation at day 2 of differentiation of KO erythroblasts cultured in 0.1 mM serine or glycine containing differentiation medium (n = 3 per group, mean + SEM). 3P glycerate, 3-phosphoglycerate; 3P pyruvate, 3-phosphohydroxypyruvate; GSH, glutathione; PE pyruvate, phosphoenolpyruvate; PPP, pentose phosphate pathway; TCA cycle, tricarboxylic acid cycle.