TRB3 induction via EPOR alleles, and TRB3-mediated survival effects in EPO-dependent erythroid progenitor cells. (A) Kit^posCD71^highTer119^neg erythroblasts were expanded, and purified from marrow progenitors as prepared from wt-EPOR, EPOR-H, and EPOR-HM mice. Cells then were cultured for 5 hours in 50 μg/mL transferrin, 0.1% BSA, 0.1 mM 2-MF, 15 ng/mL insulin, IMDM and subsequently exposed to EPO (2.5 U/mL). At the intervals indicated, cell lysates were prepared and analyzed for TRB3 expression via Western blotting. Outcomes for the wt-EPOR are illustrated in panel Ai, and for EPOR-HM and EPOR-H alleles in panel Aii. (B) TRB3 inhibits apoptosis due to cytokine withdrawal in EPO-dependent UT7epo cells. Lentiviruses encoding TRB3 or GFP only (empty control vector) were prepared and used to stably transduce UT7epo cells. GFP^pos cells were then isolated by FACS, and possible effects of TRB3 on survival were assessed in the presence of EPO at limiting doses. As assayed via staining with APC–annexin-V, significant protection against apoptosis was afforded by TRB3. Representative flow cytometry data are shown (left panels) together with mean TRB3 survival effects (± SE, n = 3). Mean outcomes for 2 independent sets of experiments also are shown (botom panel).