Stage E2 proerythroblasts deploy BCL2, whereas BCL-XL expression is activated subsequently at stage E3. (A) Stage E2 proerythroblasts (but not E1 or E3 cells) express elevated BCL2. Stage E1, E2, and E3 cells were purified from primary SP34-ex cultures, and lysates were directly prepared. Levels of BCL2 and BCL-XL were then assayed by Western blotting. (B) BCL-XL and BCL-2 expression in stage E1, E2, and E3 BM erythroid cohorts. Stage E1, E2, and E3 cells were isolated and cultured for 6 hours in the absence of hematopoietic cytokines. Cells were then exposed to EPO (5 U/mL) for 2.5 and 7.5 hours. Cell lysates were prepared and analyzed for BCL-XL, BCL-2, and β-tubulin expression via Western blotting (left panel). Band intensities of BCL-XL and BCL2 were determined using ImageJ software Version 1.42j (right panel). (C) EPO/EPOR signals regulate Bcl-xL (but not Bcl2) in late-stage E3 erythroblasts. Purified primary BM stage E1, E2, and E3 cells were cultured for 6 hours in the absence of hematopoietic growth factors and then challenged with EPO. At 30, 90, and 270 minutes, cells were lysed in Trizol, and RNA and cDNA were prepared. Bcl-xL and Bcl2 levels then were determined via quantitative RT-PCR (with beta-actin as an internal control). (D) Stage-predominant EPO induction and up-modulation of select EPO response factors, such as S3G and Trib3. Assays of transcripts were performed via quantitative RT-PCR. Values are mean ± SE.