STAT5-induced erythroid differentiation is GATA1 dependent. (A) Schematic overview of previously obtained results caused by active STAT5 overexpression. (B) Retroviral and lentiviral constructs used in this study. (C) Quantitative RT-PCR results of CB CD34⁺ cells that were double transduced with the STAT5A-ER vector in combination with the indicated lentiviral RNAi constructs. Cells were left untreated or stimulated for 24 hours with 4-OHT, sorted, and RNA was extracted. GATA1 mRNA expression was calculated after normalization against expression of hypoxanthine phosphoribosyl transferase, RPL27, and RPL30 mRNA. The graph represents the mean of 3 independent experiments. (D) Western blot of sorted cells, transduced and treated as in panel C. Extracts were blotted against STAT5, GATA1, and actin to show equal loading. (E) Representative FACS histograms of GPA expression on cells transduced with STAT5A-ER and the indicated lentiviral vectors expressing short hairpins against luciferase or GATA1. Cells were cultured on MS5 stromal cells, left untreated or stimulated with 4-OHT, and analyzed for GPA expression after 10 days. (F) MGG-stained cytospin preparations of suspension cells of cultures described in panel E and harvested after 2 weeks. (G) BFU-E numbers of methylcellulose culture of CB cells, transduced with lentiviral vectors for expression of short hairpins against luciferase and GATA1, together with an empty expression vector or a vector expressing RNAi resistant, myc-tagged GATA1. A total of 1000 double-transduced cells were sorted 1 day after transduction directly into CFC medium. BFU-E colonies were counted after 2 weeks.