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Results: 6

1.
Figure 6.

Figure 6. From: c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

Notch signaling up-regulates c-myc in normal thymocytes at the DN3 stage. Sorted DN3 thymocytes from three to five mice were incubated for 16 h on OP9 or OP9-DLL1 feeder cells in the presence of the GSI compound E (1 μM) or DMSO vehicle. Murine T6E T-ALL cells cultivated in the presence or absence of GSI for 24 h served as positive and negative controls. c-Myc transcript levels were determined by qPCR in these cells and in freshly sorted DN3a and DN3b thymocytes. Expression of c-myc was determined in three independent experiments. Mean expression levels ± 1 SD are shown.

Andrew P. Weng, et al. Genes Dev. 2006 August 1;20(15):2096-2109.
2.
Figure 3.

Figure 3. From: c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

c-Myc is necessary and sufficient to rescue T6E cells from withdrawal of Notch1 signals. (A) c-Myc rescues growth. T6E cells, which express a membrane-tethered form of Notch1 that requires cleavage by γ-secretase for activation, were transduced with MigRI retroviruses expressing GFP alone (MigRI) or GFP and the indicated polypeptides, and then treated with GSI (1 μM compound E) for up to 6 d. The numbers of GFP+ cells at each time point are shown. (B) c-Myc restores cell cycle kinetics. The DNA content of T6E cells transduced with the indicated MigRI retroviruses was measured by flow cytometry at baseline and after 6 d of treatment with GSI. (C) Inhibitors of c-myc function prevent ICN1 from rescuing T6E cells treated with GSI. T6E cells were cotransduced with retroviruses expressing ICN1 and either A-max or mad1. Cells were then cultured in the presence of 1 μM compound E for 6 d. Cells transduced with ICN1 alone (top left quadrants) are enriched over the course of the experiment, while cells expressing either A-Max or Mad1 alone (bottom right quadrants) or one of these two c-myc inhibitors and ICN1 (top right quadrant) are selected against.

Andrew P. Weng, et al. Genes Dev. 2006 August 1;20(15):2096-2109.
3.
Figure 5.

Figure 5. From: c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

Activated Notch1 rescues the T-ALL cell line 8946 from withdrawal of transgenic c-myc and conveys sensitivity to Notch pathway inhibitors. (A) Basal 8946 cell growth is not affected by treatment with GSI (1 μM compound E). (B,C) Transduction of 8946 cells with retroviruses encoding two different forms of activated Notch1, ICN1, or ΔE retroviruses, rescue the cells from the effects of doxycycline (20 ng/mL), whereas empty MigRI does not. 8946 cell rescue was judged variously by forward and side scatter (B, which shows the rescue by ICN1), or by the number of GFP positive cells on day 6 post-treatment (C, which shows the rescue by ΔE). (D) When rescued with ΔE from doxycycline treatment, 8946 cells become newly sensitized to GSI. 8946 cells transduced with ΔE were treated for 6 d with doxycline (Dox, 20 ng/mL) ± 1 μM compound E (GSI). Effects of GSI treatment were assessed by flow cytometry. (E) 8946 cells rescued from doxycycline (Dox, 20 ng/mL) by ΔE up-regulate endogenous c-myc and other Notch and c-myc target genes. 8946 cells transduced with MigRI or MigRI-ΔE were treated with doxycline (20 ng/mL) ± GSI (1 μM compound E) for 24 h Expression of deltex1 (a gene that is a up-regulated by Notch1), transgenic human c-myc, endogenous murine c-myc, and the c-myc target cad were monitored by RT–PCR.

Andrew P. Weng, et al. Genes Dev. 2006 August 1;20(15):2096-2109.
4.
Figure 4.

Figure 4. From: c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

Transduction of c-myc leads to variable rescue of human Notch-sensitive T-ALL cell lines from withdrawal of NOTCH signals. Rescue of growth (A), cell cycle progression (B), and cell size (C) were assessed in control T-ALL cell lines or the same lines transduced with c-myc and a GFP marker (c-myc) or GFP only (GFP). Cells were treated with DMSO vehicle (mock) or 1 μM compound E (GSI) for the indicated time periods. (A) The fraction of live GFP+ cells was monitored by flow cytometry. An increasing GFP+ fraction indicates a growth/survival advantage conferred by the retrovirus over untransduced GFP cells. (B) The DNA content of control cell lines was compared with that of sorted populations of cells transduced with c-myc by staining with Hoechst 33342 followed by flow cytometry. The DNA histograms shown were derived from >20,000-gated events. (C) Cell size in c-myc transduced cells and untransduced control cells treated with GSI (compound E, 1 μM) or DMSO vehicle (V) for the indicated time was determined by forward scatter of live cells. The relative cell sizes for each population (normalized to a value of 100 for vehicle-treated, untransduced control cells ± 1 SD) are depicted. The decreases in size upon addition of GSI in untransduced cells (*), and the increases in size upon transduction with c-myc in GSI-treated cultures (**, as compared with untransduced GSI-treated cells), were both significant (P < 0.01) in all cell lines tested, as measured by the Bonferroni post-test after two-way ANOVA (unweighted means) analysis.

Andrew P. Weng, et al. Genes Dev. 2006 August 1;20(15):2096-2109.
5.
Figure 1.

Figure 1. From: c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

Withdrawal of Notch1 signals down-regulates c-myc expression in T-ALL cells. (A) Identification of Notch1-sensitive genes in T6E cells. Columns represent experimental samples, while rows represent genes. Each colored box indicates relative expression level (normalized for each gene), where red indicates high and blue indicates low. The seven columns on the left are samples from T6E cells with active Notch signaling, and the six columns on the right are samples from T6E cells in which Notch signaling was inhibited. Expression data from 82 genes significantly correlated with the Notch “on” (left) versus “off” (right) distinction (p < 0.05) are depicted; expression of the 25 genes in the upper cluster increased on inhibition of Notch signaling, expression of the 57 in the lower cluster decreased. Genes implicated in the literature as related to Notch signaling as well as the novel gene c-myc are highlighted. Sample label key: (GSI [1 μM compound E] + ICN1) sorted ICN1 transduced cells treated with GSI; (GSI mock) DMSO vehicle-treated cells; (GSI 0 h) untreated cells; (GFPposA/B) sorted GFP-only cells; (GFPnegA/B) sorted untransduced cells from the same cultures as transduced cells; (GSI) cells treated with GSI for the indicated number of hours; (DN-MamA/B) sorted dominant-negative MAML1 transduced cells. (B) Northern blot analysis demonstrating the down-regulation of c-myc following treatment with GSI (1 μM compound E). Notch-dependent T6E cells and human T-ALL cell lines were treated with carrier (DMSO) or GSI for the indicated time periods. Blots were hybridized with probes indicated in the right margin.

Andrew P. Weng, et al. Genes Dev. 2006 August 1;20(15):2096-2109.
6.
Figure 2.

Figure 2. From: c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.

c-Myc is a direct target of Notch1. (A) c-Myc up-regulation by Notch does not require de novo protein synthesis. KOPT-K1 cells were treated with GSI (1 μM compound E) for 48 h to permit accumulation of the γ-secretase substrate NTM*. Cells were then washed and refed medium containing GSI (mock washout), or medium lacking GSI (washout) with or without 20 μM cycloheximide (CHX). c-Myc RNA levels were determined after 4 h of additional culture by qPCR. Each sample was assayed in triplicate; error bars correspond to standard deviations. Similar results were obtained in four independent experiments. (B) Notch1 stimulates c-myc transcription. Nuclei were prepared from KOPT-K1 cells treated with vehicle (0.01% DMSO) for 48 h; treated with GSI (1 μM compound E) for 48 h; treated with GSI for 48 h, then washed thrice and cultured 2 h in fresh medium without GSI (washout); or treated with GSI for 48 h, then washed three times and cultured 2 h in fresh medium with GSI (mock washout). RNAs isolated from runoff reactions were hybridized to slot-blots containing probes specific for c-myc and GAPDH. Bound radioactivity was quantified using a PhosphorImager. (Top panel) Phosphorimages. (Bottom panel) Calculated c-myc/GAPDH transcript ratios. Results of a representative experiment are shown. (C) Notch1 binds to the c-myc promoter through a region containing a conserved CSL consensus sequence. Chromatin immunoprecipitates were performed on cross-linked fragmented DNAs prepared from T6E cells treated with DMSO or 1 μM compound E (GSI) for 24 h. Eluted DNAs were then analyzed by qPCR performed with primers flanking putative CSL-binding sites A and B. The amount of DNA amplified from immunoprecipitated DNAs was normalized to that amplified from input DNA. (D) CSL binds to the putative target sequence in the c-myc promoter. Oligonucleotides labeled with carboxyfluorescein (FAM) were mixed with buffer alone or buffer containing purified CSL and Notch1 polypeptides. Following electrophoresis in 10% native gels, fluorescently labeled probes were detected with an 860 Storm FluorImager (Amersham Pharmacia Biotech).

Andrew P. Weng, et al. Genes Dev. 2006 August 1;20(15):2096-2109.

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