Affymetrix array–based discovery of cell-cycle–associated EPO response genes in bone marrow–derived Kit^posCD71^high erythroblasts. (A) Basic steps involved in the discovery of EPO-modulated cell-cycle regulatory genes. Erythroblasts were expanded from the bone marrow of n = 4 replicate mice. For each preparation, purified Kit^posCD71^high cells were subdivided and cultured for 6 hours in the absence of hematopoietic cytokines. Erythroblasts were then exposed to EPO (± 5 U/mL) for 90 minutes and RNA was isolated directly. Biotin-cRNA next was prepared and was used in Affymetrix 430–2.0 array profiling. Transcripts for cell-cycle–associated genes that were modulated approximately 2-fold or more by EPO (with high statistical significance) were then considered. (B) For replicate Affymetrix array data sets, and as analyzed quantitatively (mean modulation due to EPO ± SD), EPO proved to significantly modulate 9 cell-cycle–related genes: Cyclin-D2/Ccnd2, Cyclin-G2/Ccng2, Gspt1, Nupr1, Egr1, Nab2, Myc, p27/Cdkn1b, and Bcl6. (C) In quantitative RT-PCR analyses, EPO regulation of each of the EPO-modulated cell-cycle regulatory genes was confirmed. Values are means and SD for samples (and erythroblasts) from n = 3 mice.