Fas is functional in immature erythroblasts in the absence of high levels of Epo. (A) Peripheral CD34-derived day-7 and day-14 erythroblasts were incubated for 24 hours with or without 200 ng/mL of agonistic anti-Fas MoAb in the absence (−Epo) or in the presence (+Epo) of 3 U/mL recombinant Epo. Apoptosis was quantitated by DNA staining and flow cytometry analysis. (B) Day-7 cells were treated as described above with different concentrations of Epo. Percentage of protection was calculated by comparison with cells stimulated in the absence of Epo. A representative experiment out of five performed with cells from different donors is shown. (C) Lysates from day-7 and day-14 cells untreated (control) or stimulated for different times with agonistic anti-Fas MoAb (anti-Fas) were analyzed for their ability to cleave the fluorogenic caspase substrate DEVD-AFC. Data are expressed in arbitrary fluorescence units and show a representative experiment out of four performed with cells from different donors.