PHOSPHO1 depletion impairs human erythropoiesis in vitro. (A) Human CD34⁺ cells were differentiated using a 5-stage in vitro culture system and metabolites were extracted and analyzed at the end of the indicated stages of differentiation. Relative amounts of polar metabolites in the 5 groups of cells are shown in colored boxes on the right and their VIP scores are shown on the bottom. Phosphocholine and choline are labeled in bold on the left (n = 3 per group). (B) Human PHOSPHO1 gene expression at each differentiation stage normalized to 18S rRNA (n = 3 per group, mean + SEM). (C) Increased phospho-T172-AMPKα signal in PHOSPHO1-depleted human erythroblasts after 20 days of in vitro culture. Relative phospho-AMPK to AMPK signal ratio is depicted below. (D) Lower terminal proliferation rate in human erythroblasts depleted of PHOSPHO1 by transfection with 2 shRNAs. Cell number was counted after GFP⁺ cells were sorted at day 11 and at the end of Diff3, Diff4, and Diff5 (n = 3 per group, mean ± SEM). (E) Lower percentages of enucleation in PHOSPHO1-depleted human erythroblasts after 20 days of in vitro culture. CD34⁺ cells were expanded and differentiated as described in “Materials and methods.” Enucleation as judged by Hoechst staining and glycophorin A surface expression was measured at day 20. (F) Control and PHOSPHO1-depleted human erythroblasts were cultured in medium supplemented with addition of 0.1 mM glycine or 0.2 mM serine at day 12. Cell numbers were counted at day 12 and day 16; fold cell expansion is shown. (G) hPHOSPHO1 RNA expression in control and shRNA knocked-down cells that were assayed in panels D through F (n = 3 per group, mean + SEM).