Ikaros expression during cytokine induced erythroid differentiation. (A) Highly purified CD34⁺ cells from human CB or from human adult mPB samples were maintained in conditions that drive differentiation to the erythoid lineage. On day 3, cells were transduced with EF1 GFP or EF1 IK6 vectors. Cultures were stopped on day 20 for CB samples, and on day 17 for mPB samples. On days 10, 13, 17, and 20, erythroblast subsets derived from CD34⁺ CB cells were identified by May-Grünwald-Giemsa staining. Results are expressed as the mean percentage in 7 independent experiments. (B) On days 10, 13, 17, and 20, total RNA was extracted from cultured erythroid cells in CB samples, and expression of endogenous Ikaros isoforms was visualized by ethidium bromide staining of 2% agarose gel after RT and 40 cycles of amplification of cDNA with primers designed to match exons 2 and 7 (Ikaros isoforms in Table S1) in order to amplify all Ikaros isoforms. Arrows indicate the positions of Ik1-, Ik2/3-, and Ik4-expressed isoforms. (C) On days 10, 13, 17, and 20, total RNA was also extracted from adult cultured erythroid cells. Expression of Ikaros in CB was quantified using quantitative PCR and normalized relatively to endogenous GAPDH gene expression. Results are shown with means (± SEM) for 4 to 7 independent CB samples.