GATA-2 deficiency induces PU-1/Sfpi1 and reprograms G1ME cells to macrophages. (A) Relative expression of Gata2 and selected myeloid and macrophage-specific target genes in G1ME cells transduced with Banshee control (C) or Banshee shGATA2 retrovirus and flow-purified by GFP positivity. Mean plus or minus standard deviation values are shown for one representative experiment performed in triplicate. CCAAT/enhancer binding protein alpha (Cebpa), myeloperoxidase (Mpo), colony-stimulating factor receptor 1 (Csfr1), and macrophage 1 (Mac1). (B) Morphology of G1ME cells transduced with control or shGATA2 virus 6 days after infection. The shG2-infected cells are larger with abundant cytoplasm-containing granules and vacuoles. May-Grünwald Giemsa stain. Original magnification, ×63. Photographs were obtained using an Axioskope 2 microscope equipped with an AxioCam camera and AxioVision acquisition software (Carl Zeiss Microimaging) at room temperature. (C) Representative flow cytometric analysis of Banshee control– and Banshee shGATA2–infected G1ME cells. The numbers indicate the percentage of GFP⁺ cells within the live population in the top panels and the percentage of Mac-1⁺ F4/80⁺ within the GFP⁺ population in the bottom panels. (D) Macrophage stimulation assays of Banshee control (C)– and Banshee shGATA2–transduced G1ME cells. Tumor necrosis factor (TNF) and nitric oxide (NO) levels were measured from the supernatant of unstimulated (US) cells or 18 hours after stimulation with 100 ng/mL lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ). GATA-2 knockdown G1ME cells induced TNF secretion and produced nitric oxide at baseline and after stimulation with LPS and IFN-γ. Mean ± SD values are shown for one representative experiment performed in triplicate.