Gene array–based discovery of Epo-modulated cytokines and cell-surface adhesion factors in murine bone marrow–derived erythroblasts. (A) Illustrated are steps used to expand and isolate Kit⁺CD71^highTer119⁻ erythroblasts together with a representative flow cytometric profile. (B) For erythroblasts prepared from independent bone marrow preparations (n = 4), hematopoietic cytokines were withdrawn for 6 hours and cells were then exposed to Epo (± 5 U/mL). At 90 minutes, RNA was prepared and used in transcriptome analyses. As a control, levels of Epo-induced Cis1 transcript levels (right panel) were analyzed by quantitative RT-PCR. (C) For Epo-regulated genes, genome-wide outcomes are illustrated by relative difference analyses. Cel files were analyzed using ChipInspector. Significantly changed probes were then defined by SAM analysis (left panel) at a false discovery rate of 0.0% (and corresponding significantly changed transcripts needed to be covered by at least 3 such probes). The SAM analysis method is described by Tusher et al⁶⁹  and Storey and Tibshirani.⁷⁰  The false discovery rate is a measure for discrimination of significant features. (D) Affymetrix 430–2.0 array-based analyses of Epo-regulated cytokine and cell-surface adhesion factors. Values are mean fold-modulation by Epo (± a standard error [SE], n = 4). (E) Quantitative RT-PCR analyses of Epo regulation of OncoM, Gdf3, Cxcr4, Itga4, and PODXL. Values are means (± SE) and are normalized to beta-actin. (F) For PODXL, Cxcr4, and Itga4, cell-surface levels were also assayed (by flow cytometry) among Kit⁺CD71^high erythroblasts following their isolation and subsequent 24-hour culture in Epo at 0.1, 0.4, and 1.6 U/mL in the presence or absence of SCF (50 ng/mL).