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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
Files in this Data Supplement:
- Figure S5. Altered gene expression pattern and Mib1 deletion are evident in MMTV-Cre;Mib1f/f mice (JPG, 82.1 KB)
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RT-PCR analysis of cDNA from whole BM of 14- to 19-week-old MMTV-Cre;Mib1+/f (WT) and MMTV-Cre;Mib1f/f (MT) mice with the indicated genes. Myeloid-affiliated genes (M-CSFR, G-CSFR, GM-CSFR, and MPO), lymphoid-affiliated genes (Aiolos and IL-7Rα), erythroid-affiliated genes (β-globin and GATA-1), leukemia-associated transcriptional factors (PU.1, ICSBP, Ikaros, and AML-1b), and Mib1 were analyzed. β-actin was used for normalization.
- Figure S6. Increased numbers and cycling rate of blood and BM cells in MMTV-Cre;Mib1f/f mice (JPG, 61.7 KB)
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(A–B) Flow cytometric analysis of blood (A) or BM cells (B) from 13- to 15-week-old MMTV-Cre;Mib1+/f (WT) and MMTV-Cre;Mib1f/f (MT) mice, which were injected i.v. with BrdU (1 mg) 6 hr before sacrifice. (C) The expression of Bclxl in the CD11b+Gr-1+ granulocytes sorted from 15- to 19-week-old WT and MT splenocytes. β-actin was used as a loading control.
- Figure S7. Increased numbers of LSK and GMP were dependent on Mib1-null microenvironment (JPG, 61.2 KB)
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(A–B) Flow cytometric analysis of BM cells from reciprocal BMT recipient mice. A distribution of LSK (A) and GMP (B) were shown. Numbers indicate the representative percentage of described populations in total BM (n=6).
- Figure S8. Both MMTV-Cre;Rosa-Notch1 and MMTV-Cre;Mib1f/f;Rosa-Notch1 mice develop transplantable T-cell leukemia (JPG, 131 KB)
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(A) Splenomegaly in the 34-week-old MMTV-Cre;Mib1f/f;Rosa-Notch1 (right) compared to an age-matched Mib1f/f;Rosa-Notch1 mouse as a control (left). (B–F) Flow cytometric analysis of BM cells from 34- to 35-week-old MMTV-Cre;Rosa-Notch1, MMTV-Cre;Mib1f/f;Rosa-Notch1, and control mice. A representative cell size distribution of BM cells (B–C). Note that MMTV-Cre;Mib1f/f;Rosa-Notch1 BM cells have large cell sizes (FSChi) and that GFP-positive cells express CD5 (upper panel), CD3 (middle panel), and CD8 (bottom panel) (D). Cell cycle distribution of whole BM cells (E) and GFP+ and GFP− BM cells (F). (G) A representative hematoxylin and eosin staining of kidney, lung, and liver from MMTV-Cre;Rosa-Notch1 mice. Note that there are massive leukocyte infiltrations. (H) Flow cytometric analysis of the BM cells from the reconstituted MMTV-Cre;Mib1f/f and MMTV-Cre;Mib1+/f mice transplanted by sorted GFP-positive BM cells from MMTV-Cre;Rosa-Notch1 mice.
- Figure S9. Intact trabecular bone and osteoblasts in MMTV-Cre;Mib1f/f mice (JPG, 133 KB)
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Histological analysis of the tibias from 15- to 20-week-old MMTV-Cre;Mib1+/f (WT) and MMTV-Cre;Mib1f/f (MT) mice injected with 5-FU (250 mg/kg) three times for 12 days. The trabecular bone (A) and the bone-lining osteoblasts (B, arrows) are shown. Asterisk (*) indicates adipocytes in the BM.
- Figure S10. More than 99% of BM cells from the reconstituted mice were of donor origins (JPG, 57.7 KB)
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(A–C) Flow cytometric analyses of BM cells in the reconstituted mice. Lethally irradiated CD45.2 MMTV-Cre;Mib1f/f (MT) mice (A and B) and CD45.2 MMTV-Cre;Mib1f/f;Rosa-Notch1 (MT;N1ICD) mice (C) were injected i.v. with CD45.1 congenic BM cells. Six (A and B) or seven (C) weeks after transplantation, the reconstituted BM cells were analyzed by flow cytometry.
- Figure S11. Abnormal structures and decreased numbers of adipocytes in MMTV-Cre;Mib1f/f BM microenvironment (JPG, 138 KB)
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(A) Hematoxylin and eosin staining (×400; inset ×200) of the bones from 15- to 20-week-old MMTV-Cre;Mib1+/f (WT) and MMTV-Cre;Mib1f/f (MT) mice. Note that adipocytes (arrowhead) were evident in WT bone, but not in MT bone, and that abnormal structures like a spongy (*) were seen only in MT bone. (B and C) Immunohistochemical analysis of the spleens from 15- to 20-week-old WT and MT mice. Reticulin staining (×400; inset ×100) (A). SMA (in green) and OPN (in red) double immunohistochemistry (×200) (B). A representative of three independent experiments is shown. (D) Real-time RT-PCR analysis of Rb transcripts in whole BM cDNAs from 16- to 20-week-old WT and MT mice. Scale bars: 50 µm (A and B); 100 µm (C); 100 µm, Scale bars in the inset of (A); 200 µm, Scale bars in the inset of (B).
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