STAT5-induced block in myeloid differentiation is reversed after GATA1 depletion. (A) CD34⁺ CB cells were transduced with STAT5A-ER and the indicated lentiviral RNAi constructs. After transduction, cells were cultured on MS5 stromal cells and either left untreated or stimulated with 4-OHT. Each week, half of the medium was removed and double-transduced cells were sorted and cytospin preparations were made (left panel). The same cells were analyzed by flow cytometry for the expression of CD11b and GPA at weeks 1, 2, 3, and 5. Representative FACS plots are shown in the right panel with percentage positive cells indicated in the plots. (B) FACS analysis of the expression of GPA, CD11b, CD14 (monocyte/macrophage), CD15 (granulocyte), and CD71 (transferrin receptor) of cells cultured and stimulated as in panel A at week 2. (C) FACS data as in panel B from week 5. (B-C) One representative experiment of 3 experiments performed is shown. (D) CD34⁺ CB cells were transduced with the indicated lentiviral shRNA constructs. One day after transduction, the cells were stained for CD34, CD38, CD45RA, and CD123 and transduced (EGFP-positive) cells were sorted according to the shown gates to obtain MEP and GMP progenitor populations. The sorted cells were cultured in methylcellulose medium to enumerate the number of BFU-Es and CFU-GMs. After 2 weeks, progenitors were scored. (E) GATA1 expression in GMPs and MEPs as determined by quantitative RT-PCR and Illumina arrays.