notch1 c terminal domain  (R&D Systems)

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) Luciferase reporter assays of SupT1 T-ALL cells cotransfected with a reporter construct containing a genomic CD44 region (–4.8 to –3.5 kb) spanning 5 putative CSL-binding sites along with ICN1-encoding retrovirus plus or minus retrovirus encoding dnMAML1 or with GFP-only control. Data are represented as mean fold induction ± SEM over luciferase activity of control cells cotransfected with an empty reporter vector (pGL3) and the GFP-only vector (n = 3). s1–s5, CSL-binding sites 1–5. (B) Representative ChIP assay performed either with anti-NOTCH1 Abs (anti-cleaved NOTCH1, for CUTLL1 and HPB-ALL, or anti–C-terminal NOTCH1, for SupT1) or with the corresponding control Ab. PCR amplification was done on input and immunoprecipitated DNA with primer pairs spanning each of the 5 CD44 CSL sites or the reported c-MYC CSL-binding site. The vertical dividing line separates lanes run noncontiguously on the same gel. (C) Quantitative data of ChIP assays in B. Results are shown as fold enrichment of anti-NOTCH1–immunoprecipitated DNA over DNA immunoprecipitated with control Abs. Bars represent mean values ± SEM (n = 3). Ctrl, control. (D) Luciferase reporter assay of SupT1 T-ALL cells cotransfected with a reporter vector containing the indicated combinations of WT and mutated (mut) sequences corresponding to the 5 putative CSL-binding sites identified in CD44, along with either retrovirus encoding ICN1 together or not with dnMAML1-encoding retrovirus or with GFP-only retrovirus as control. Data are represented as mean fold induction SEM over control cells as in A (n = 3). Data shown in A and D were corrected by FDR. (E) Chromatin landscapes upstream of the CD44 locus were derived from ChIP-seq raw data of human CUTLL1 cells under steady-state (basal), Notch-off, and Notch-on conditions (GEO GSE51800), previously described by Wang et al. (23). The presence of a 5′ distal enhancer containing a dynamic NOTCH1-PBPJ site and a RUNX1 site is shown. H3K27ac peaks associated with this dynamic NOTCH1 site are shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: ChIP assays were performed as described ( 19 ) using polyclonal Abs against either the C-terminal NOTCH1 domain (Santa Cruz Biotechnology Inc.) or cleaved NOTCH1 (Val1744) (Cell Signaling Technology).

Techniques: Luciferase, Construct, Binding Assay, Control, Activity Assay, Plasmid Preparation, Amplification, Immunoprecipitation, Reporter Assay, Derivative Assay, ChIP-sequencing

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A and B) CB HSPCs transduced with ICN1 and GFP (7.4% ± 1.7%) were transplanted into NSG mice. Combined data from 4 independent experiments, with a total of 18 mice, are shown as percentages of human cells derived from nontransduced ICN1– (A) or ICN1-transduced (B) HSPCs engrafting the mouse BM at the indicated months after transplant. (C) Kaplan-Meier survival curves of mice shown in A and B. (D) Absolute numbers of human ICN1+ cells infiltrating the BM, spleen, thymus, and liver of diseased mice shown in B at 7 months after transplant. Data were normalized to 105 transduced input cells. Mean values combined from 4 independent experiments with 5 to 6 mice/group are shown. (E) Mean percentages ± SEM of human ICN1+ cells of the indicated cell subsets, as defined in Figure 1C, infiltrating the BM, spleen, and liver of diseased mice shown in B (n ≥ 3). (F) Representative phenotype of human ICN1+ cells infiltrating the BM and spleen of diseased mice shown in B (n ≥ 7). (G) Representative CD44 expression of human DP T cells derived from ICN1-transduced or nontransduced CB HSPCs, infiltrating the indicated organs of diseased mice shown in B (n ≥ 7). (H) Reverse-transcriptase PCR (RT-PCR) analysis of the TCR-Vβ repertoire of 3 samples of human ICN1+ DP T cells infiltrating the BM of diseased mice shown in B (bottom panels) at 7 months after transplant (n = 8). The polyclonal repertoire of DP thymocytes isolated from human postnatal thymus is shown as control (upper panel). ****P < 0.0001.

Article Snippet: ChIP assays were performed as described ( 19 ) using polyclonal Abs against either the C-terminal NOTCH1 domain (Santa Cruz Biotechnology Inc.) or cleaved NOTCH1 (Val1744) (Cell Signaling Technology).

Techniques: Transduction, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: The Journal of Experimental Medicine

Article Title: CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

doi: 10.1084/jem.20081922

Figure Lengend Snippet: Notch1 signaling regulates IL-7Rα expression in human hematopoietic precursors. (A) Flow cytometry of IL-7Rα expression on electronically gated GFP + CD4 + CD8 + (DP) thymocytes derived from ETPs transduced with either ICN1-GFP or GFP-only vectors in an FTOC assay (day 13). (B) IL-7Rα expression on primary CD34 + CB progenitors (left) and on electronically gated GFP + Lin − progenies, derived from CD34 + progenitors transduced with either ICN1-GFP or GFP-only vectors and cultured with multilineage-supportive cytokines for 15 d (right). (C) IL-7Rα expression levels on electronically gated GFP + Lin − progenies derived from CD34 + progenitors transduced with either dnMAML1-GFP or GFP-only vectors and cocultured on OP9-DL1 stroma for 22 d. Shaded histograms represent background staining with irrelevant isotype-matched antibodies. Numbers in quadrants represent MFI values (A) and percentages of positive cells (B and C). Results are representative of at least three independent experiments.

Article Snippet: Polyclonal antibodies against either the C-terminal domain of Notch1 or PU.1 (Santa Cruz Biotechnology, Inc.) were used to label Notch–DNA or PU.1–DNA complexes.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Transduction, Cell Culture, Staining

Journal: The Journal of Experimental Medicine

Article Title: CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

doi: 10.1084/jem.20081922

Figure Lengend Snippet: CSL/MAML-mediated transcriptional activation of IL7R by active Notch1. (A) Identification of a conserved CSL-binding site in the 5′ regulatory region of human IL7R and mouse Il7ra . Numbers indicate distances in base pairs from the transcription initiation site. (B) Luciferase reporter assays in 293 T cells cotransfected with a reporter construct containing the 5′ regulatory region of IL7R shown in A, along with either a retroviral vector encoding ICN1 and GFP (ICN1) or a GFP-only control vector (GFP). Data are represented as fold induction over luciferase activity of control cells cotransfected with an empty reporter vector (pGL3B) and the GFP-only vector. (C) MAML-dependent activation of IL7R transcription. Reporter assays were performed in Jurkat cells cotransfected with the IL7R reporter and ICN1-GFP, and with or without dnMAML1-GFP. Data are represented as fold induction over luciferase activity of control cells transfected with the GFP-only vector. (D and E) Notch-induced IL7R promoter activity requires an intact CSL-binding site. Reporter assays were performed in RBP-Jκ +/− and RBP-Jκ −/− MEFs cotransfected with the IL7R reporter along with either ICN1-GFP or GFP-only vectors (D) and 293 T cells cotransfected with ICN1-GFP along with a reporter vector containing either the wild-type sequence of the CSL-binding site in the IL7R promoter or the mutated (mut) CSL sequence shown in A (E). Bar graphs represent means ± SEM of triplicate samples from at least four independent experiments. (F) ICN1 binds to the IL7R promoter in vivo. Formaldehyde cross-linked chromatin from primary DN2 human thymocytes, SupT1 pre-T cells and CUTLL1 T-lineage cells, and REH, NALM-6, and HPB-NULL pre-B cells was subjected to ChIP with specific antibodies against human Notch1 (N1; top), or PU.1 (bottom). Goat or rabbit Igs were used, respectively, as controls. PCR was done on input DNA and on immunoprecipitated DNA with primers pairs spanning the CSL sites of HES1 and IL7R (top) or the Ets site of IL7R (bottom). Results are from one representative out of two to three independent experiments.

Article Snippet: Polyclonal antibodies against either the C-terminal domain of Notch1 or PU.1 (Santa Cruz Biotechnology, Inc.) were used to label Notch–DNA or PU.1–DNA complexes.

Techniques: Activation Assay, Binding Assay, Luciferase, Construct, Retroviral, Plasmid Preparation, Control, Activity Assay, Transfection, Sequencing, In Vivo, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

doi: 10.1084/jem.20081922

Figure Lengend Snippet: Notch1 regulates IL-7Rα expression and IL-7–dependent proliferation in T-ALL. (A) Surface IL-7Rα expression analyzed by flow cytometry on DND41, HPBALL, and CUTLL1 T-ALL cell lines. Background fluorescence (shaded) was determined with an irrelevant isotype-matched antibody. (B) Percentages of IL-7Rα–expressing T-ALL cells cultured with the GSI CompE were determined by flow cytometry and normalized to 100% IL-7Rα + cells recovered from DMSO-treated controls at the indicated times. (C) Relative IL-7Rα expression levels on CUTLL1 cells transduced either with a retrovirus encoding IL-7Rαand GFP or with a GFP-only vector were determined by flow cytometry upon culture with either GSI CompE or DMSO for 10 d. MFI values were normalized to IL-7Rα expression values on GFP-transduced CUTLL1 cells treated with DMSO. (D) Relative protein level (left) and function (right) of IL-7Rα receptors expressed on CUTLL1 cells transduced and cultured as in C were determined by flow cytometry after surface staining of IL-7Rα and intracellular staining of phosphorylated STAT5 with specific mAbs. Background fluorescence (shaded) was determined with irrelevant isotype-matched mAbs. (E) Relative numbers of cells in S-G2-M phases of cell cycle from a representative experiment in (C) were determined on gated GFP + - and IL-7Rα + –transduced CUTLL1 cells by day 18. (F) Percentages of GFP + and IL-7Rα + CUTLL1 cells transduced and cultured as in C were normalized to 50% of transduced cells at day 0. Data in B, C, and F are means ± SEM of at least three independent experiments. Results in D and E are from one of three independent experiments performed on different days.

Article Snippet: Polyclonal antibodies against either the C-terminal domain of Notch1 or PU.1 (Santa Cruz Biotechnology, Inc.) were used to label Notch–DNA or PU.1–DNA complexes.

Techniques: Expressing, Flow Cytometry, Fluorescence, Cell Culture, Plasmid Preparation, Staining