At a Kit^negCD71^high stage of development, Lyn^-/- erythroid progenitor cells fail to efficiently enter an apparent G1/S cell cycle phase and undergo increased apoptosis. (A) Cultures of expanded Lyn^+/+ and Lyn^-/- erythroblasts were analyzed for Kit and CD71 marker expression, and were costained for DNA content with DRAQ5. DRAQ5 staining distributions are shown for total cell populations (i), for Kit^negCD71^high cells (ii), and for Kit^posCD71^high cells (iii). Note the limited frequencies of Kit^negCD71^highLyn^-/- erythroblasts in the S-phase peak compared directly with Lyn^+/+ controls (arrows, ii). (B) Increased apoptosis of Lyn^-/- erythroblasts at a Kit^negCD71^high stage. Bone marrow-derived Lyn^-/- and wild-type control erythroblasts were cultured in SP34-EX (with SCF and Epo at nonlimiting concentrations). At 120 hours of culture, cells were coanalyzed for Kit (CD117) and CD71 expression, and for annexin V positivity. Selectively within a Kit^negCD71^high population, Lyn^-/- cells exhibited significantly increased levels of annexin V staining (33.4% vs 14.2%, or 220% over Lyn^+/+ controls). This is illustrated in a representative flow cytometric profile (i) and in histograms as mean values (± SE) for triplicate analyses (ii) (results shown are representative of 2 independent experiments). (C) Also analyzed (by Western blotting) were levels of GATA-1 expression (and GAPDH) in Kit^posCD71^high as well as Kit^negCD71^highLyn^-/- and Lyn^+/+ erythroblasts (as isolated by MACS).