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Blood, Vol. 112, Issue 12, 4628-4638, December 1, 2008
Defective Notch activation in microenvironment leads to myeloproliferative disease
Blood Kim et al.
112: 4628
Supplemental materials for: Kim et al
Both MMTV-Cre;Rosa-Notch1 and MMTV-Cre;Mib1f/f;Rosa-Notch1 mice develop transplantable T-cell leukemia
MMTV-Cre;Mib1f/f;Rosa-Notch1 mice also develop the same T-cell leukemia as MMTV-Cre;Rosa-Notch1 mice (Fig. S8). They exhibited typical splenomegaly (Fig. S8A) and blast cells with large cell sizes (Figs. S8B and S8C). The blast cells in the BM from both MMTV-Cre;Rosa-Notch1 and MMTV-Cre;Mib1f/f;Rosa-Notch1 mice were positively marked by CD5, CD3, and CD8, as well as GFP indicative of Cre-mediated recombination (Fig. S8D). More than 70% of BM cells were arrested in the G0/G1 phases of the cell cycle, with less than 30% in the S and G2/M phases in control mice, however, more than 50% of BM cells were in the S and G2/M phases in MMTV-Cre;Mib1f/f;Rosa-Notch1 mice (Fig. S8E). Particularly, about 70% of GFP positive blast cells among BM cells were in the S and G2/M phases, while majority of GFP negative BM cells were arrested in the G0/G1 phases of the cell cycle in MMTV-Cre;Mib1f/f;Rosa-Notch1 mice, indicating that only the cells with Cre-mediated recombination became autonomously leukemic (Fig. S8F). The blast cells massively infiltrated into multiple organs (Fig. S8G), and when they were transplanted to lethally-irradiated wild-type and MMTV-Cre;Mib1f/f mice, both reconstituted mice developed donor originated T-cell leukemia (Fig. S8H).
Files in this Data Supplement:
- Table S1. Bone marrow cellularity and hepatosplenomegaly (PDF, 15 KB)
- Figure S1. Inactivation of Mib1 in both the whole BM cells and the BM stromal cells, in which CD45+ and CD11b+ cells were depleted, from MMTV-Cre;Mib1f/f mice (JPG, 66.1 KB)
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The expression of Mib1 in the whole BM cells (A) and the cultured primary BM-derived, CD45+ and CD11b+ cells depleted, stromal cells (BM-SCs) (B and C) from 15- to 19-week-old MMTV-Cre;Mib1+/f (WT), MMTV-Cre;Mib1f/f (MT) mice, Cell lysates were blotted with anti-Mib1/Dip1 antibody. βActin was used for normalization. The relative level of expression of Mib1 was designated as 100% (C).
- Figure S2. Extensive extramedullary hematopoiesis in the spleens from MMTV-Cre;Mib1f/f mice (JPG, 111 KB)
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(A–B) Flow cytometric analysis of the splenocytes from 15-week-old MMTV-Cre;Mib1+/f (WT) and MMTV-Cre;Mib1f/f (MT) mice. A representative distribution of LSK (Lin−Sca-1+c-Kit+), MP (Lin−Sca-1−c-Kit+) (A), and erythrocytes (CD71+Ter119+) (B) (n=7). (C) Immunohistochemistry of spleens from 15- to 20-week-old WT (left) and MT mice (right). TER119 (in green), DNA (Hoechst, in blue). Note that the large erythroblasts with nuclei (arrowheads), as compared to the mature erythrocytes (arrows). Scale bar: 50 µm. (D) Platelet counts of peripheral bloods from 14- to 16-week-old WT (n=6) and MT (n=16) mice. Bars indicate the mean.
- Figure S3. Enhanced granulocytosis in the BM, spleen, lymph node, and thymus of MMTV-Cre;Mib1f/f mice (JPG, 117 KB)
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Flow cytometric analysis of the splenocytes (A and C), BM cells (B and F), lymph node cells (D), and thymocytes (E) from 15- to 20-week-old MMTV-Cre;Mib1+/f (left) and MMTV-Cre;Mib1f/f (right) mice. Numbers indicate the mean ± SD (n=5).
- Figure S4. Splenomegaly in MMTV-Cre;Mib1f/f mice (JPG, 147 KB)
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(A–B) Immunohistochemical analysis of the spleens from 15- to 20-week-old MMTV-Cre;Mib1+/f (WT) and MMTV-Cre;Mib1f/f (MT) mice. Reticulin staining (×400; inset ×100) (A). MPO (in green) and PCNA (in red) double immunohistochemistry (×200) (B). A representative of three independent experiments is shown. Scale bars: 50 µm (A); 100 µm (B); 100 µm, Scale bars in the inset.
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