VEGF-A165 enhances the expression of bothζ and ϵ embryonic globins. (A-B) RT-PCR analyses of the embryonic (ϵ and ζ) and adult (β) globins compared in EBs differentiated for 15 days in the presence of cytokines+BMP-4 (-V) or cytokines+BMP-4+VEGF-A165 (+V) (A) and in their derived erythroid colonies aspirated from methylcellulose after 15 days of culture (B). Human fetal head cDNA (pos) was used as a positive control. Because no products were detectable in the absence of the reverse transcriptase (no RT), only one “no RT” reaction is shown in (A). β-actin PCR product signal intensity was used for standardization and relative quantitation. (C-D) Time course of the intracellular expression performed by flow cytometry of the HbF globin in EBs differentiated as indicated (C) and of both HbF and the HbA globins in the derived erythoid colonies (D). Results are expressed as the mean percentages of positive cells, with the corresponding SD (vertical bars) (n = 2 to 4 independent experiments in each condition). (E) RT-PCR analysis of GATA-1, PU-1, and SCL/Tal-1 transcription factors in EBs differentiated as indicated and described above (n = 3 independent experiments for each condition).
