4-Hydroxy-tamoxifen-induced Stat5a-ER* activation replaced Epo in erythropoiesis. (A) Scheme of the 4-OH-T-inducible Stat5-ER* constructs used, encoding fusion proteins of WT Stat5a, cS5, or Stat5aΔ749 with ER* (see “Methods”). (B) Western blot analysis of phosphorylated Stat5 (P-Y-Stat5), and total Stat5 protein in WT fetal liver erythroblasts expressing GFP or Stat5a-ER*. The larger protein recognized by P-Y-Stat5 and Stat5 antibodies corresponds to Stat5a-ER*. Actin, loading control. (C) Cumulative cell numbers (1 representative experiment of 3) of proliferating primary erythroblast cultures expressing Stat5a-ER* or GFP determined in the presence of Epo (+ EPO, −4-OH-T, left, normal self-renewal conditions), the presence of 4-OH-T (5 nM) instead of Epo (−Epo, + 4-OH-T, middle) and without Epo and 4-OH-T (right). (D) Percentage of apoptotic cells of cultures in panel C at day 6 (arrows in panel C) as analyzed by annexin V staining. (E) WT fetal liver cells expressing Stat5a-ER* or GFP were subjected to CFU-E assays in the presence of Epo (left panels) or 4-OH-T (50 nM) instead of Epo (right panels). Acid benzidine–positive colonies were scored at day 2. (F) Cytospins of cells retrieved from the CFU-E assays in panel E and stained with hematoxylin/eosin and for hemoglobin (brownish color).