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Blood, Vol. 113, Issue 19, 4702-4710, May 7, 2009
Enhanced expression of p210BCR/ABL and aberrant expression of Zfp423/ZNF423 induce blast crisis of chronic myelogenous leukemia
Blood Miyazaki et al.
113: 4702
Supplemental materials for: Miyazaki et al
Files in this Data Supplement:
- Table S1. Genes identified by iPCR in p210BCR/ABL/BXH2 lymphoid leukemic samples (PDF, 20.9 KB)
- Figure S1. Flow cytometric analysis of CD19-positive leukemic cells (JPG, 192 KB)
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CD19-positive samples (No.17, 18, and 30) were stained with antibodies against CD20, B220, BP-1, CD43, and IgM. The profile of IgM and CD43 were gated on CD19-positive cells. The histgarms show BP-1 or CD20 expression on CD19-positive cells (black line) compared with negative control cells (gray filled). All the samples were positive for CD20, B220, BP-1, CD43 but negative for IgM, indicating that they were pre–B-cell leukemias.
- Figure S2. Southern blot analysis for rearrangement of immunoglobulin heavy chain region (JPG, 37.9 KB)
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Genomic DNAs extracted from a control p210BCR/ABL transgenic mouse spleen (C) and tumor tissues of four leukemic mice expressing B-cell surface markers (No. 17 and No.30; transgene integration, and No.18,and No.23; Zfp423 integration) were digested with EcoRI and blotted with mouse JH probe. In the tumor DNA samples, rearranged bands (indicated by arrowheads) are observed in addition to the germline band (G).
- Figure S3. Quantitation of p210BCR/ABL mRNA expression (JPG, 54.1 KB)
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mRNAs extracted from a wild-type mouse spleen (WT), a control transgenic mouse spleen (C), and tumor tissues of four leukemic mice (No. 17 and No.30; transgene integration, and No.18,and No.23; Zfp423 integration) were subjected to real-time quantitative PCR for p210BCR/ABL expression. The mRNA expression levels are shown as a relative amount to that of the control transgenic mouse spleen (C).
- Figure S4. Cell proliferation assay of a p210BCR/ABL-positive, ZNF423–non-expressing line, KOPN67 (JPG, 22 KB)
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1 × 105 cells of the parental KOPN67 line and two shRNAs-introduced sublines (KOPN67/shRNA-1 and KOPN67/shRNA-2) were plated on day 1 and cell numbers were counted on day 3 and day 5. The mean cell number of three independent experiments of each line is plotted with error bars.
- Figure S5. Western blot for retrovirus-mediated expression of p210BCR/ABL and Zfp423 (JPG, 45.1 KB)
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Proteins extracted from 3T3 cells tranfected with pMysIG and pMysIG/p210BCR/ABL were blotted with an anti-ABL antibody (left panel) and those from 3T3 cells tranfected with pMysIKO and pMysIKO/FHZfp423 were blotted with an anti-HA antibody (right panel). The positions of p210BCR/ABL and FHZfp423 proteins are indicated by arrows and protein markers are shown on the left.
- Figure S6. Expression of p210BCR/ABL and FHZfp423 mRNAs in tumors developed in mice reconstituted with BM cells transduced with p210BCR/ABL and FHZfp423-expressing retroviruses (JPG, 44.9 KB)
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RNAs extracted from tumors of 9 leukemic mice (2 CML, 5 B-ALL, and 2-AML cases) were subjected to RT-PCR for p210BCR/ABL and Zfp423. Result of β-actin RT-PCR is shown as an internal control.
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