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Blood, Vol. 109, Issue 6, 2356-2364, March 15, 2007
Gfi1b:green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1
Blood Vassen et al.
109: 2356
Supplemental materials for: Vassen et al, Vol 109, Issue 6, 2356-2364
Files in this Data Supplement:
- Figure S1. Gfi1b expression correlates with development but not with activation (JPG, 69.1 KB)
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(A) Spleen cells from Gfi1b+/GFP mice were cultured in RPMI (+10 mM HEPES, + 1 mM sodium pyruvate) and treated with lipopolysaccharide (LPS), erythropoietin (EPO), or PBS as a control as indicated (rightmost) for 24 hours. Nonadherent living cells were stained for the surface markers B220, Ter119, and CD86 and analyzed by FACS. LPS-induced activation of B220+ cells is shown in the histogram (upper middle) showing induction of the activation marker CD86. GFP expression of the same populations was analyzed (overlay histogram, upper right panel). GFP expression of all living cells is shown in the lower middle histogram. Note, that only after treatment with EPO a GFP-high population proliferates. The erythroid origin of this population was verified by electronic gating for Ter119-positive cells and measurement of GFP expression in these cells. The respective histograms are shown in the lower right panel. (B) Autoregulatory features of Gfi1b were analyzed in bone marrow cells from crossbreeds of Gfi1b+/GFP mice with vav-Gfi1b transgenic mice.15 Whole bone marrow cells (WBCs) from wt (black), wt/KI (Gfi1b+/GFP, light grey), and wt/KI/tg (Gfi1b+/GFP/Gfi1b-tg, dark grey) mice were stained for B220 expression and living cells were analyzed for GFP expression. Histograms for GFP expression in WBCs (left), B220-negative WBCs (middle), and B220high cells of these mice were shown. Note that only in B220high cells a significant down-regulation by the transgene is detectable. (C) Overlay-histograms showing GFP expression in B cells and T cells from wt and Gfi1b+/GFP measured by FACS mice after specific activation. Spleen cells from wt and Gfi1b+/GFP mice were isolated and seeded in RPMI (+10 mM HEPES + 1 mM sodium pyruvate) on 48-well plates uncoated or coated with anti-CD40 or anti-CD3 antibodies and treated with IL4 (5 ng/mL) or Concancavalin-A (Con-A; 2.5 µg/mL) for 44 hours. Nonadherent cells were stained for B220 or CD3 expression and electronically gated cells were analyzed for GFP expression (control indicates treatment with PBS).
- Figure S2. Lack of Gfi1b expression in memory T cells from spleen (JPG, 62.8 KB)
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(A) CD4+ T cells (left) from spleens of adult wt or wt/KI (Gfi1b+/GFP) mice were electronically gated for CD44 and CD62L expression (middle), and GFP expression of the respective subpopulations is shown in histograms (right). (B) Same as in panel A but for CD8+ T cells. Note that no GFP expression is detected in memory T cells.
- Figure S3.The expression of Gfi1 and Gfi1b throughout the hematopoietic compartment (JPG, 47.7 KB)
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Histograms show roughly the relative expression of Gfi1 (gray) and Gfi1b (black) in the different cell types. (The expression pattern of Gfi1 in granulocytes and monocytes is preliminary data according to Dr. Hui Zeng (Institute of Infectious disease, Beijing Ditan Hospital, Beijing, P.R. China; oral communication). HSC indicates hematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte/macrophage progenitor; MEP, megacaryocyte/erythrocyte progenitor; T-L, T lymphocyte; B-L, B lymphocyte; Gran, granulocyte; Mon, monocyte; Ery, erythocyte; Meg, megakaryocyte.
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