Bone marrow erythroblast preparations, EPO-dependent survival responses, and transcriptome-based identification of EPO-regulated survival genes. (A) Bone marrow preparations were expanded for 3 days in SP34-EX medium to yield Kit^posCD71^highTer119^neg, Kit^negCD71^highTer119^neg, and Kit^negCD71^highTer119^pos erythroblast populations. Each was then purified by MACS and/or FACS, and increases in survival as afforded by EPO were determined. Specifically, cells were cultured in SP34-EX medium in the absence of SCF and presence of EPO at 0.1 U/mL. At 18 hours, frequencies of apoptotic cells were assayed using FITC–annexin-V and flow cytometry. Values are normalized means plus or minus SE. Kit^posCD71^highTer119^neg erythroblasts as purified by multiparameter MACS also were visualized in cytospin preparations and were assayed for protection against programmed cell death at a range of EPO doses. In the lower left panel, flow cytometry analyses also depict the homogeneity of isolated Kit^posCD71^highTer119^neg cells. (B) Kit^posCD71^highTer119^neg erythroblasts were cultured for 6 hours in the absence of hematopoietic cytokines, and were then exposed to EPO (± 5 U/mL) for 90 minutes. This included parallel processing of bone marrow–derived erythroblasts from n = 4 independent mice. After EPO exposure, RNA was isolated directly; 4 μg was used in biotin-cRNA syntheses and Affymetrix 430-2.0 array hybridizations. Among candidate (anti)apoptosis-related genes, 8 proved to be modulated by EPO at a confidence interval more than 99%. This included Irs2, Trb3, Trb2, Foxo3a, Bim, Pim1, Pim3, and Serpina3G (S3G). Values are mean-fold modulation plus or minus SD.