Figure 1
Figure 1. Cul4A+/− HSCs exhibit an engraftment defect. (A) Expression of Cul4A protein in Lin− LDMNCs. LDMNCs were isolated from Cul4A+/− and wild-type littermates, and Lin− cells were obtained by negative selection (Lineage Cell Depletion Kit; Miltenyi Biotec, Auburn, CA). Cell lysates were prepared and analyzed by immunoblot as described.12,13 Quantitation of scanned film was performed using ImageJ (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD) and normalized for β-actin loading. (B) Competitive repopulation with a 1:1 ratio of test donor cells and competitors; overall percentage donor chimerism. For Cul4A+/− or wild-type donors (C57BL/6), 5 × 105 bone marrow LDMNCs (CD45.2+) were mixed with 5 × 105 wild-type competitor cells (B6.BoyJ, CD45.1+) and transplanted into 5 to 6 lethally irradiated wild-type recipients (CD45.1+) by tail vein injection. Overall engraftment (percentage CD45.2+) for Cul4A+/− (■) or wild-type (□) test cells was measured 2 to 6 months after transplantation by flow cytometric analysis of peripheral blood with anti-CD45.1 or anti-CD45.2 antibodies conjugated with fluorescein isothiocyanate (FITC; Pharmingen, San Diego, CA). (C) Engraftment into myeloid and lymphoid lineages. For the transplantation described in panel B, engraftment of Cul4A+/− or wild-type test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was also measured using biotin-conjugated anti-Gr-1, -Mac-1, -B220, or both -CD4 and -CD8 antibodies, respectively, and then streptavidin-APC secondary antibody (Pharmingen), each in combination with the anti-CD45.1 and -CD45.2 antibodies described for panel B. Percentage CD45.2+ for Cul4A+/− (■) or wild-type (□) test cells is graphed for each lineage 6 months after transplantation. (D) Competitive repopulation with a 1/3:1 ratio of test donor cells and competitors; overall percentage donor chimerism. For Cul4A+/− or wild-type donors, 1.7 × 105 bone marrow LDMNCs were mixed with 5 × 105 wild-type competitors in a competitive repopulation analogous to that described for panel B. Overall engraftment was determined in the same way, and graphed for Cul4A+/− (■) or wild-type (□) test cells 2 to 6 months after transplantation. (E) Engraftment into myeloid and lymphoid lineages for test cells mixed 1/3:1 with competitors. For the competitive repopulation described in panel D, percentage CD45.2+ for Cul4A+/− (filled bars) or wild-type (open bars) test cells was determined as described in panel C and graphed for each lineage 6 months after transplantation. For panels B-E, mean (± SEM) is graphed. (F) Limiting dilution. From 5 × 103 to 2 × 105 Cul4A+/− bone marrow LDMNCs and 5 × 103 to 5 × 104 wild-type cells were each mixed with 5 × 105 wild-type competitors and transplanted into recipients (6-7 recipients per each test cell type and amount, except only 3 recipients for 5 × 104 wild-type cells and 9 recipients for 2 × 105 Cul4A+/− cells). The percentage of recipients not engrafted (overall percentage donor chimerism less than 5%) was plotted versus test cell dose. The 5% cutoff was chosen, because in control transplantations where only C57Bl/6J wild-type cells (CD45.2+) were transplanted into B6.BoyJ (CD45.1+) recipients, the background host percentage chimerism was approximately 5% 6 months after transplantation.

Cul4A+/− HSCs exhibit an engraftment defect. (A) Expression of Cul4A protein in Lin− LDMNCs. LDMNCs were isolated from Cul4A+/− and wild-type littermates, and Lin− cells were obtained by negative selection (Lineage Cell Depletion Kit; Miltenyi Biotec, Auburn, CA). Cell lysates were prepared and analyzed by immunoblot as described.12,13  Quantitation of scanned film was performed using ImageJ (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD) and normalized for β-actin loading. (B) Competitive repopulation with a 1:1 ratio of test donor cells and competitors; overall percentage donor chimerism. For Cul4A+/− or wild-type donors (C57BL/6), 5 × 105 bone marrow LDMNCs (CD45.2+) were mixed with 5 × 105 wild-type competitor cells (B6.BoyJ, CD45.1+) and transplanted into 5 to 6 lethally irradiated wild-type recipients (CD45.1+) by tail vein injection. Overall engraftment (percentage CD45.2+) for Cul4A+/− (■) or wild-type (□) test cells was measured 2 to 6 months after transplantation by flow cytometric analysis of peripheral blood with anti-CD45.1 or anti-CD45.2 antibodies conjugated with fluorescein isothiocyanate (FITC; Pharmingen, San Diego, CA). (C) Engraftment into myeloid and lymphoid lineages. For the transplantation described in panel B, engraftment of Cul4A+/− or wild-type test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was also measured using biotin-conjugated anti-Gr-1, -Mac-1, -B220, or both -CD4 and -CD8 antibodies, respectively, and then streptavidin-APC secondary antibody (Pharmingen), each in combination with the anti-CD45.1 and -CD45.2 antibodies described for panel B. Percentage CD45.2+ for Cul4A+/− (■) or wild-type (□) test cells is graphed for each lineage 6 months after transplantation. (D) Competitive repopulation with a 1/3:1 ratio of test donor cells and competitors; overall percentage donor chimerism. For Cul4A+/− or wild-type donors, 1.7 × 105 bone marrow LDMNCs were mixed with 5 × 105 wild-type competitors in a competitive repopulation analogous to that described for panel B. Overall engraftment was determined in the same way, and graphed for Cul4A+/− (■) or wild-type (□) test cells 2 to 6 months after transplantation. (E) Engraftment into myeloid and lymphoid lineages for test cells mixed 1/3:1 with competitors. For the competitive repopulation described in panel D, percentage CD45.2+ for Cul4A+/− (filled bars) or wild-type (open bars) test cells was determined as described in panel C and graphed for each lineage 6 months after transplantation. For panels B-E, mean (± SEM) is graphed. (F) Limiting dilution. From 5 × 103 to 2 × 105Cul4A+/− bone marrow LDMNCs and 5 × 103 to 5 × 104 wild-type cells were each mixed with 5 × 105 wild-type competitors and transplanted into recipients (6-7 recipients per each test cell type and amount, except only 3 recipients for 5 × 104 wild-type cells and 9 recipients for 2 × 105Cul4A+/− cells). The percentage of recipients not engrafted (overall percentage donor chimerism less than 5%) was plotted versus test cell dose. The 5% cutoff was chosen, because in control transplantations where only C57Bl/6J wild-type cells (CD45.2+) were transplanted into B6.BoyJ (CD45.1+) recipients, the background host percentage chimerism was approximately 5% 6 months after transplantation.

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