Transient expression of PU Transient expression of PU.1 commits multipotent progenitors to a myeloid fate whereas continued expression favors macrophage over granulocyte differentiation  Zoe McIvor, Sibyll Hein, Heike Fiegler, Timm Schroeder, Carol Stocking, Ursula Just, Michael Cross  Experimental Hematology  Volume 31, Issue 1, Pages 39-47 (January 2003) DOI: 10.1016/S0301-472X(02)01017-2

Figure 1 Effect of constitutive PU.1 expression on proliferation and colony formation. Proliferation (A) and colony-forming efficiency (B) of mixed populations of FDCPmix cells transfected with PU.1-encoding (rPU.1neo) or control (rneo) retrovirus. (C) Control colonies show the compact structure typical of undifferentiated cells, whereas those derived from PU.1-expressing cells show the dissipate morphology indicative of myeloid differentiation. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)

Figure 2 PU.1 expression levels and proliferation rates of individual clones. Western blot of whole-cell extracts from both mixed populations and single clones of FDCPmix transfected with PU.1-expressing (rPU.1neo) and control (rneo) retrovirus. After PU.1 detection, the blot was reprobed with an antibody to actin to confirm equal loading. The corresponding proliferation rates of each clone are shown below the Western blot as a histogram of cumulative cell number over a 4-week growth period. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)

Figure 3 Transcriptional activation of the cfms promoter by transiently transfected PU.1. Luciferase expression driven by the PU.1-responsive cfms promoter construct transfected either alone or in combination with plasmids directing the expression of GFP (pCMVExp3eGFP) or of PU.1 plus GFP (pCMVPU.1eGFP). Means and standard deviations of triplicate transfections within a single experiment are shown. The experiment was repeated twice with essentially the same results. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)

Figure 4 FACS of productively transfected cells. Example of the FACS profiles of FDCPmix transiently tranfected with the PU.1/GFP coexpression vector pCMVPU.1.eGFP. Sixteen hours after electroporation and immediately after removal of dead cells by Ficoll gradient centrifugation, GFP-expressing cells were identified in dot plots of green (530/30 nm filter) vs red (630/22 nm filter) fluorescence. To maximize the purity of the sorted cell populations, the fluorescent cells initially were isolated using the enrichment mode, and the enriched population subsequently sorted at high resolution. Profiles of the initial, enriched, and sorted populations are shown. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)

Figure 5 Effects of transient PU.1 expression on colony-forming efficiency. The colony-forming efficiency of control FDCPmix (Untr.) and FACS-sorted cells expressing GFP alone (GFP+ cont.) or GFP plus PU.1 (GFP+ PU.1). GFP− PU.1 denotes nonfluorescent cells sorted from the same transfected population as the GFP+ PU.1 sample. Results are given as means and standard deviations of triplicate colony assays from a single experiment. The experiment was repeated three times with essentially the same results. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)

Figure 6 Effects of transient PU.1 expression on growth factor dependence. Growth of FDCPmix cells transiently transfected with plasmids expressing GFP alone (pCMVExp3eGFP) or PU.1 plus GFP (pCMVPU.1eGFP). Productively transfected cells were FACS sorted 12 hours after electroporation, then replaced immediately into media supporting self-renewal (A), erythroid differentiation (B), or granulocyte macrophage differentiation (C). Values are given as means and standard deviations of cumulative cell numbers per milliliter in triplicate cultures from one of three experiments. The experiment was repeated twice with the same results. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)

Figure 7 Effects of PU.1 on granulocyte/macrophage differentiation. Morphologic analysis of the differentiated progeny of transfected cells cultured under conditions that support terminal granulocyte macrophage differentiation of FDCPmix. (A) Stable transfectants harboring either the control rneo (Cont.) or the rPU.1 neo (PU.1) vector. Mix = nonclonal populations. (B) FDCPmix cells transiently transfected with PU.1-expressing (pCMVPU.1eGFP) and control (pCMVExp3eGFP) plasmids. sr = Self-renewal medium containing 150 U/mL IL-3. gm, g, and m = media containing 2 U/mL IL-3 plus GM-CSF, GM-CSF + G-CSF, and GM-CSF + M-CSF, respectively. Values shown are means of duplicate or triplicate cultures from individual experiments. Each experiment was repeated at least twice with the same results. May-Grünwald-Giemsa staining of undifferentiated (C) and differentiated (D) pCMVPU.1eGFP transfected cells is shown to illustrate the morphologic features of differentiation (magnification ×40). Undifferentiated blast cells are of moderate size with a high nuclear-to-cytoplasmic ratio and dark cytoplasmic staining. Granulocyte differentiation results in a reduction in cell size, lighter-staining cytoplasm, and a characteristic segmented or ring-form nucleus. Monocyte/macrophage cells have a large cytoplasmic volume, lighter-staining cytoplasm, and round or indented nuclei. Experimental Hematology 2003 31, 39-47DOI: (10.1016/S0301-472X(02)01017-2)