Heme metabolism pathway analysis during normal human erythroid differentiation. (A) Measurement of total heme content from day 4 to day 14. Quantification based, after iron removal, on the protoporphyrin quantification by direct molecule fluorescence. Mean OD relative to that obtained at day 4 (mean OD = 1). The data are mean ± standard deviation of 3 independent experiments. (B) Expression levels of the major proteins involved in heme synthesis (ALAS1, ALAS2, FECH), excess free heme removal (FLVCR1, BACH1, HMOX1), globin transcription (GATA1), globin translation (HRI, EIF2α, α and β globins, some RP involved as well in DBA, RPS19, RPL5, RPL11), and iron uptake (TfR1). Immunoblots of 50 000 human normal primary erythroid cells during the normal erythroid culture time course from day 0 to day 14 (0, 2, 4, 7, 9, 11, and 14) obtained from healthy CD34⁺ cord blood. Proteins were compared with β-actin expression. (C) Protein expression of ALAS2, FECH, BACH1, FLVCR1, EIF2α, and α and β globins relative to β-actin at days 0, 2, 4, 7, 9, 11, and 14 of normal erythroid differentiation. *P < .05, **P < .010.