American Society of Hematology

Figure 3.

$Figure 3. GATA-1 induces erythromegakaryocytic maturation of G1ME cells. (A) Retroviral constructs used for gene rescue. The MIGR1 vector encodes green fluorescent protein (GFP) linked to an internal ribosome entry site (IRES). MIGR1-GATA-1 also contains the full-length coding region of murine GATA-1 cDNA. For panels B to E, cells were analyzed 3 days after retroviral transduction. Viral infection efficiencies assessed by flow cytometry for GFP expression were about 65% for MIGRI and 35% for MIGR1-GATA-1. (B) GATA-1 protein expression in transduced cells determined by Western blotting. The amount of whole-cell lysate analyzed in each lane is indicated. MEL cell lysate was analyzed in parallel for comparison (last 3 lanes). (C) Expression of the erythroid-specific surface marker Ter119. Percentages in panels refer to fraction of GFP+ cells expressing Ter119. Approximately 65% of MIGR1- and 30% of GATA-1-transduced cells were GFP+. (D) Expression of the terminal megakaryocyte maturation marker GPIb. Percentages in panels refer to fraction of GFP+ cells expressing GPIb. (E) Morphology of cells after GATA-1-induced maturation. The top panels show benzidine staining for hemoglobin, with dark brown benzidine-positive cells visible only in the GATA-1-rescued sample. From the flow cytometry results for Ter119 in panel 3C, roughly 6% of cells are expected to be benzidine positive. The bottom panels show May-Gr̈nwald-Giemsa (MGG) staining with large multinucleated megakaryocytes occurring specifically in the GATA-1-rescued sample. Original magnification, × 400. Photographs were taken by using a microscope (Axioskop 2; Carl Zeiss) equipped with a color digital camera (Axiocam; Carl Zeiss).$

GATA-1 induces erythromegakaryocytic maturation of G1ME cells. (A) Retroviral constructs used for gene rescue. The MIGR1 vector encodes green fluorescent protein (GFP) linked to an internal ribosome entry site (IRES). MIGR1-GATA-1 also contains the full-length coding region of murine GATA-1 cDNA. For panels B to E, cells were analyzed 3 days after retroviral transduction. Viral infection efficiencies assessed by flow cytometry for GFP expression were about 65% for MIGRI and 35% for MIGR1-GATA-1. (B) GATA-1 protein expression in transduced cells determined by Western blotting. The amount of whole-cell lysate analyzed in each lane is indicated. MEL cell lysate was analyzed in parallel for comparison (last 3 lanes). (C) Expression of the erythroid-specific surface marker Ter119. Percentages in panels refer to fraction of GFP⁺ cells expressing Ter119. Approximately 65% of MIGR1- and 30% of GATA-1-transduced cells were GFP⁺. (D) Expression of the terminal megakaryocyte maturation marker GPIb. Percentages in panels refer to fraction of GFP⁺ cells expressing GPIb. (E) Morphology of cells after GATA-1-induced maturation. The top panels show benzidine staining for hemoglobin, with dark brown benzidine-positive cells visible only in the GATA-1-rescued sample. From the flow cytometry results for Ter119 in panel 3C, roughly 6% of cells are expected to be benzidine positive. The bottom panels show May-Gr̈nwald-Giemsa (MGG) staining with large multinucleated megakaryocytes occurring specifically in the GATA-1-rescued sample. Original magnification, × 400. Photographs were taken by using a microscope (Axioskop 2; Carl Zeiss) equipped with a color digital camera (Axiocam; Carl Zeiss).

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