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Blood, Vol. 110, Issue 10, 3753-3762, November 15, 2007
NOTCH1 pathway activation is an early hallmark of SCL T leukemogenesis
Blood Göthert et al.
110: 3753
Supplemental materials for: Gothert et al
Files in this Data Supplement:
- Document 1. Supplemental methods and references (PDF, 33.1 KB)
- Figure S1. Characterization of tamoxifen-inducible lck-ERT2-SCL transgenic mice (JPG, 116 KB)
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(A) lck-ERT2-SCL transgenic construct. In order to establish a conditional transgenic model of SCL induced T-ALL we created a fusion-protein between SCL and the tamoxifen (TAM)-responsive ligand-binding domain of the human estrogen receptor (ERT2).1 We aimed to minimize steric hindrance between ERT2 and SCL by placing the SCL cDNA 3′ of ERT2 and inserting an intervening linker-peptide (amino acid sequence: GGGGSGGGG).6,7 T-cell specific expression of the generated ERT2-SCL cDNA was achieved by guiding expression with the lck proximal promoter.5 The locations of primers (triangles, P1 and P2) and oligo probe (OP, line) used for RT-PCR expression analysis are shown. (B) Schematic drawing of the transgenic lck-ERT2-SCL system. The lck-ERT2-SCL transgene drives early T-cell specific expression of the ERT2-SCL fusion protein. TAM treatment leads to translocation of ERT2-SCL from the cytoplasm to the nucleus, where SCL presumably discloses its oncogenic potential. (C) Thymic expression of the lck-ERT2-SCL transgene was confirmed by RT-PCR with transgene specific, intron spanning primers (P1 and P2 in (A)). A transgene specific, spliced 392 base pair (bp) product (592 bp, size of genomic product) was detected by agarose gel analysis (upper panel) in thymi from mice of transgenic founder lines A and B. The product sequence was confirmed by hybridizing the blotted gel (lower panel) with a transgene-specific oligo probe (OP in (A)). (D) In order to confirm TAM-dependent nuclear translocation of ERT2-SCL we compared anti-ER antibody immunostained frozen sections of thymi from lck-ERT2-SCL mice with and without TAM treatment. As expected, sections of lck-ERT2-SCL thymi without TAM treatment showed a honeycomb-like staining pattern: the cytoplasm of thymocytes stained positive for ER leaving the nucleus devoid of staining. In contrast, sections of lck-ERT2-SCL thymi following administration of TAM showed strong ER-staining of the nucleus with rather weak cytoplasmic staining. This demonstrated that the location of the ERT2-SCL fusion protein could be controlled within lck-ERT2-SCL thymocytes by the administration of TAM. Indicated transgenic and littermate control mice were treated with a tamoxifen-containing feed (1 g/kg) and control feed, respectively, for four weeks before thymus harvest. Representative sections of founder line ‘B’ are shown. Scale bar indicates 50 µm. (E) Functional analysis of lck-ERT2-SCL transgenic founder lines was performed by analyzing fetal thymic organ cultures (FTOC). It has been described by others that constitutive aberrant SCL-expression leads to an altered CD4/CD8 ratio within the thymus.8 Thus, we analyzed fetal lck-ERT2-SCL and control thymi for the expression of CD4 and CD8 after seven days of culture in the presence TAM. Fetal thymi were harvested on day E16.5 and cultured in the presence of hydroxy-tamoxifen (OH-TAM, 100 nM) for 7 days before the flow cytometric analysis for CD4 and CD8 expression. Representative plots of a lck-ERT2-SCL (founder line B) and a wild-type littermate thymus are shown. The analysis revealed nuclear translocation of ERT2-SCL leading to a disturbed CD4/CD8 ratio within fetal thymi ex vivo. These data demonstrated the functionality of the newly created ERT2-SCL fusion protein. All subsequent experiments were carried out with lck-ERT2-SCL founder line ‘B’ displaying the strongest SCL-induced aberrant thymic phenotype.
- Figure S2. The perturbation of T-cell development induced by the lck-ERT2-SCL transgene is dependent on the administration of tamoxifen (JPG, 89 KB)
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Four-week-old lck-ERT2-SCL and wild-type littermates were assigned to receive either tamoxifen-containing feed (1g tamoxifen/kg; TAM feed) or control feed. After three weeks thymocytes were harvested and analyzed by flow cytometry. In the absence of tamoxifen administration the thymic immunophenotype of lck-ERT2-SCL mice was indistinguishable from the thymic immunophenotype of their wild-type littermates. Representative plots are shown. SP, single-positive; FSC, forward scatter.
- Figure S3. The HSA-expression level of tamoxifen-treated lck-ERT2-SCL CD8+TCRβlow cells is more similar to DP TCRβlow cells than to wild-type control CD8+TCRβlow cells (JPG, 41.9 KB)
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Thymocytes of tamoxifen (TAM)-treated lck-ERT2-SCL and wild-type control mice were stained for CD4, CD8, TCR and HSA (CD24) and analyzed by flow cytometry. The HSA-expression histograms of CD8+TCRlow and DP TCRlow cells are displayed comparing thymocytes from TAM-treated lck-ERT2-SCL and wild-type littermate control mice. Representative histograms of lck-ERT2-SCL (n=3) and wild-type (n=3) mice are shown.
- Figure S4. Tamoxifen feed does not alter the rate of apoptosis of normal wild-type thymocytes (JPG, 31.1 KB)
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Thymocytes were harvested from TAM-treated (9 weeks) wild-type mice and from littermate control mice receiving normal feed for the same time-period. The thymic cellularity, the percentage of dead cells (propidium iodide+, PI+) and the proportion of apoptotic thymocytes (Annexib V+/PI−) were determined. TAM, tamoxifen.
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