Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: NOTCH1 is expressed and activated in naïve and memory B cells, putative normal counterparts of CLL. (A) Gene-expression profile analysis (HG-U133 Plus 2.0 Array) of NOTCH1, MYC, HES1, and BCL6 in normal mature naive, GC, and memory B-cell subpopulations isolated from human tonsils (23). Each column corresponds to an independent sample. The mRNA expression pattern of NOTCH1 in naïve and memory B cells is similar to that of MYC, typically expressed only in a small fraction of GC–B cells (69), and opposite to that of BCL6, a known GC master regulator (81). Moreover, NOTCH1 expression levels are concordant with those of HES1, a NOTCH1 target in multiple tissue types (11). (B) Immunoblot (IB) analysis of ICN1, BCL6, MYC, and control β-actin in mature B-cell subpopulations isolated from human tonsils. (C) Immunofluorescence (IF) staining of ICN1, the dark-zone GC-marker AID (82), and the B-cell–specific surface antigen CD20 in a human tonsil section. (D) Tracking of the HALLMARK_NOTCH_SIGNALING geneset from the Molecular Signatures Database v5.1 (software.broadinstitute.org/gsea/msigdb/index.jsp) in normal mature B-cell subpopulations by GSEA. Abbreviations: DZ, dark zone; LZ, light zone; M, mantle zone.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Gene Expression, Isolation, Expressing, Western Blot, Control, Immunofluorescence, Staining, Marker, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: Primary CLL cases express ICN1 because of NOTCH1 PEST-truncations or alternative mechanisms. (A) IB analysis of ICN1 and control β-actin in 10 representative PB CLL cases, 4 carrying NOTCH1 PEST-truncations (ΔPEST) and 6 NOTCH1–wild-type (WT), in the control T-ALL cell line CUTLL1 (83) and in MO1043 CLL cells cocultured with OP9 stromal cells expressing the NOTCH1 ligand DL1 (54). The full set of analyzed primary CLL cases, including those reported here, is displayed in Fig. S2. (B) Frequency of ICN1 positivity in 124 primary CLL cases. (C) IF staining of ICN1 in primary ICN1+ (pos) and ICN1− (neg) CLL cells and in the control CUTLL1 T-ALL cell line in basal conditions (+) and upon Compound E (CpE, 24 h, 1 μM) treatment (−).

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Control, Expressing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: ICN1 expression analysis in a panel of primary CLL cases and PBMC. (A) IB analysis of ICN1 and control β-actin in a panel of 124 CLL PB primary CLL cases, (B) in primary NOTCH1–wild-type CLL cells treated with the γ-secretase inhibitor Compound E (CpE, 500 nM, 8 h) or control DMSO, and (C) in PBMC protein extracts and representative primary CLL cases expressing ICN1. Samples are color-coded based on the NOTCH1 mutational status [red, clonal NOTCH1 PEST-truncating events; orange, subclonal NOTCH1 PEST-truncating events; blue, RAG-mediated NOTCH1 translocation (83); and black, NOTCH1–wild-type]. Samples in gray were excluded from the analysis because of low quality of the protein lysate, low viability, or low leukemic representation. Color-coded arrows indicate cases subjected to RNA-Seq analysis: dark red denotes NOTCH1-mutated cases expressing ICN1; blue, NOTCH1–wild-type cases expressing ICN1; and green, ICN1− NOTCH1–wild-type cases. Abbreviations: MO+DL1, MO1043 cells cocultured on OP9-DL1 cells (54); s.e., short exposure; l.e., long exposure.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Expressing, Control, Translocation Assay, RNA Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: Identification of NOTCH1 direct targets in CLL. (A) Hierarchical clustering of RNA-Seq profiles of MO1043-ICN1-HA and -eGFP cells (Pearson correlation with average linkage, minimum log2 expression 5 and minimum SD 1). (B) Scatter plot of log2-transformed RNA-Seq FPKM values of differentially expressed genes between MO1043-ICN1-HA and -eGFP control CLL cells (FDR < 0.001). (C and D) Distribution of NOTCH1 binding sites (BS) in the genome of MO1043-ICN1-HA CLL cells. (E) Functional classification of NOTCH1-BS mapping to proximal promoters and distal regions of the genome based on their overlap with the H3K4me3, H3K4me, H3K27Ac and H3K27me3 histone marks. (F) Rank order of increasing H3K27Ac fold-enrichment at enhancer loci in in MO1043-ICN1-HA CLL cells. (G) Overlap between NOTCH1-BS and superenhancers identified with the ROSE algorithm (35, 36). (H) Representative examples of genes regulated by NOTCH1 via binding to superenhancer regions. (I) Intersection between RNA-Seq and ChIP-Seq data obtained in MO1043-ICN1-HA CLL cells. (J) Top three significantly (P = 1.00E-15) enriched transcription factor motifs lying ±200 bp of NOTCH1-BS. Abbreviations: NoExp, transcripts not expressed in MO1043-ICN1-HA cells; NoMov, transcripts not moving upon ICN1-HA expression; SEs, superenhancers; TF, transcription factor.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: RNA Sequencing, Expressing, Transformation Assay, Control, Binding Assay, Functional Assay, ChIP-sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: CLL superenhancers features. (A) Overlap of MO1043-ICN1-HA superenhancers with the H3K27me3 and H3K4me1 chromatin marks. (B) Expression levels (log2 FPKM) of genes associated with superenhancers (SE) or regular enhancers (E) identified in MO1043-ICN1-HA cells. (C) Differential up-regulation of genes associated with NOTCH1 binding sites (BS) overlapping with superenhancer regions or located elsewhere in the genome (“other”) of MO1043-ICN1-HA cells. In A and C, P values are reported according to a two-tailed Fisher’s exact test. In B, the P value was calculated based on an unpaired unequal variance two-tailed Student's t test.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Expressing, Binding Assay, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: Significant enrichment of top NOTCH1-bound genes in MO1043-ICN1-HA compared with control -eGFP cells. (A) GSEA enrichment plot depicting significant enrichment of a geneset composed by the top 400 NOTCH1-bound genes (i.e., top genes ranked based on ChIP-Seq P values) in MO1043-ICN1-HA CLL cells compared with -eGFP controls and (B) corresponding leading edge genes.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Control, ChIP-sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: The NOTCH1 CLL signature is enriched in primary CLL cases expressing ICN1. (A) GSEA enrichment plots depicting significant enrichment of the NOTCH1 CLL signature in NOTCH1-mutated (M) and wild-type (WT) primary CLL cases expressing ICN1+ (ICN1-pos) compared with ICN1− (ICN1-neg) cases, and heatmap of RNA-Seq profiles of corresponding leading edge genes (n = 90) (B).

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Expressing, RNA Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: Representative examples of NOTCH1-direct target genes. Representative ChIP-Seq plots depicting NOTCH1 binding and histone marking patterns at genes that are bound by NOTCH1 and up-regulated in MO1043-ICN1-HA CLL cells. The y axes in the ChIP-Seq plots indicate fragment density in reads per million (rpm).

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: ChIP-sequencing, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: A NOTCH1-bound superenhancer region regulating MYC expression is recurrently duplicated in CLL. (A) NOTCH1 occupancy profiles and histone marks patterns in the 8q24 region encompassing the MYC locus (chr8:128000000–129000000, hg19) in primary CLL cases and MO1043-ICN1-HA cells, with corresponding peaks depicted in the box below the ChIP-Seq plots. The y axes in the ChIP-Seq plots indicate fragment density in reads per million (rpm). The two boxes below the called peaks represent segmentation data (7, 53) visualized using IGV (2.3.59), with red denoting a region of CN gain, blue a CN loss, and white depicting a normal (diploid) CN. Individual genes in the region are aligned in the Bottom panel. (B) Schematic representation of the distribution of superenhancers, NOTCH1 binding sites, and RBPJ motifs in the 8q24 region encompassing the MYC locus. (C) In the heatmap, rows correspond to normal or malignant B cells (52, 84) and two control T-ALL cell lines, and columns represent the two superenhancers identified in the 8q24 region encompassing the MYC locus, color-coded based on their presence or absence in the displayed cell type (light gray, absent; black, present). (D) ChIP-qPCR analysis of NOTCH1 and H3K27Ac at the MYC-associated superenhancer regions identified in MO1043-ICN1-HA CLL cells; results are presented relative to those obtained with IgG (IgG; control) and to a distal actin locus, set as 1. (E) qRT-PCR analysis of MYC and HES1 mRNA expression in three representative primary CLL cases, upon NOTCH1 signaling induction via coculture on stromal OP9-DL1 cells in the presence or absence of the γ-secretase inhibitor Compound E (CpE, 24 h, 1 μM, Left and Center), or upon basal NOTCH1 signaling inhibition in the presence of CpE (Right). Results are represented relative to those of CLL cells cocultured on OP9 stromal cells (Left), on OP9-DL1 stromal cells in the presence of CpE (Center), or with vehicle DMSO (Right), set as 1. The full set of analyzed primary CLL cases, including those represented here, is displayed in Fig. S9. The bar graphs in D and E show the mean values, and the error bars represent the SD between triplicates. Abbreviations: B-LCL, B-lymphoblastoid cell line; DLBCL, diffuse large B-cell lymphoma; MCL, mantle cell lymphoma; SE, superenhancer; SLL, small lymphocytic lymphoma.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Expressing, ChIP-sequencing, Binding Assay, Control, ChIP-qPCR, Quantitative RT-PCR, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: Focal copy number gains recurrently affect the NOTCH1-bound 8q24 B-cell–specific superenhancer region in CLL. (A) Overlap between NOTCH1-bound 8q24 superenhancers observed in CLL and other tissues. In the heatmap, rows correspond to different tissues (normal and malignant) reported in the dbSUPER database (84) and columns represent the two superenhancers identified in the 8q24 region encompassing the MYC locus, color-coded based on their presence or absence in the displayed cell type (light gray, absent; black, present). (B) Frequency of NOTCH1 mutations and MYC CN gains (including gains encompassing only the MYC-associated superenhancer region) in a panel of 452 primary CLL cases, as reported in Puente et al. (7) and of MYC CN gains (including gains encompassing only the MYC-associated superenhancer region) in a panel of 353 primary CLL cases (53). (C) Graphic display of CN data from 30 patients harboring CN gains involving the 8q24 region encompassing the newly identified MYC-associated superenhancer regions in CLL. Segmentation data were visualized using IGV (2.3.59), where each track represents one sample, and white denotes a normal (diploid) CN, red a region of CN gain and blue a CN loss. Individual genes in the region are aligned in the Bottom panel, and the red boxed area highlights the minimal common region (MCR) of CN gain. In the bottom are highlighted the locations of NOTCH1 binding sites, RBPJK motifs (RBP_Jkappa V$RBPJK_Q4 and V$RBPJK_01 from the TRANSFAC database) and the superenhancers identified in CLL. (D) Heatmap showing the distribution of NOTCH1 mutations and MYC CN gains identified in n = 71/452 primary CLL cases, as reported in Puente et al. (7). In the heatmap, each column corresponds to a different case, and the two Bottom rows represent NOTCH1 mutations (M) and MYC alterations (act), color-coded based on their presence or absence in the displayed case (light gray, absent; black, present). The Top row shows the IGHV mutational status of the displayed cases (M, mutated; NA, not available; UM, unmutated).

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: MYC RNA levels are responsive to modulation of NOTCH1 signaling activation in primary CLL cases. qRT-PCR analysis of MYC and HES1 mRNAs expression in primary CLL cases upon ICN1 induction via coculture on stromal OP9-DL1 cells in the presence or absence of the γ-secretase inhibitor Compound E (CpE, 1 μM, 24 h, top five graphs), or upon basal NOTCH1 signaling inhibition in the presence of CpE (Bottom graph). Results are represented relative to those of CLL cells cocultured on OP9 stromal cells, on OP9-DL1 stromal cells in the presence of CpE, or with vehicle DMSO, set as 1. ICN1-positive cases depicted in the Bottom panel (n = 6) include 3 NOTCH1-mutated and 3 NOTCH1–wild-type cases.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

doi: 10.1073/pnas.1702564114

Figure Lengend Snippet: H3K27Ac ChIP-Sequencing plots at the NOTCH1 locus in nine primary CLL cases. The y axes in the ChIP-Seq plots indicate fragment density in reads per million. M, mutated; WT, wild-type.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anticleaved NOTCH1 (clone D3B8, Cell Signaling Technology), mouse monoclonal anti-MYC (clone 9E10, Santa Cruz), mouse monoclonal anti-BCL6 (clone GI191E/A8, Cell Marque), rabbit monoclonal anti-HA (clone C29F4, Cell Signaling Technology), mouse monoclonal anti–β-actin (clone AC-15, Sigma), rabbit polyclonal anti–β-tubulin (H-235, Santa Cruz).

Techniques: ChIP-sequencing

Journal: The Journal of Experimental Medicine

Article Title: Internal deletion of BCOR reveals a tumor suppressor function for BCOR in T lymphocyte malignancies

doi: 10.1084/jem.20170167

Figure Lengend Snippet: Activation of Myc in Bcor ΔE4/y DP thymocytes and T-ALL. (a) GSEA plot for the MYC target gene set demonstrating significant positive enrichment in Bcor ΔE4/y DP T-ALL cells relative to WT DP thymocytes. NES, NOM, and FDR are indicated. Red and blue colors represent positive (up-regulated in the given genotype relative to WT) and negative (up-regulated in WT relative to the given genotype) enrichment, respectively. (b) Quantitative RT-PCR analysis of Myc in various hematopoietic cell fractions and Bcor ΔE4/y DP T-ALL cells. Hprt1 was used to normalize the amount of input RNA. Data are shown as the mean ± SD ( n = 3). Representative data from two independent experiments are presented. (c) Scatter diagram showing RNA sequence data. Signal levels of RefSeq genes (RPKM+1 in log2) in Bcor ΔE4/y DP T-ALL cells and WT DP thymocytes are plotted. Light gray lines represent the boundaries for a twofold increase and twofold decrease. Representative direct target genes of NOTCH1 are shown as red dots. (d) Chromatogram traces showing a Notch1 mutation in exon 34 (T-ALL no. 1). The variant bases are listed underneath and indicated in gray boxes. (e) Cleaved NOTCH1 protein in T-ALL cells detected by Western blot analysis. Cleaved NOTCH1 proteins in human T-ALL cells (Jurkat) and BCOR ΔE4 T-ALL cells (CD8 SP and CD4 SP T-ALL cells from the spleens of T-ALL nos. 4 and 5, respectively) are indicated by arrowheads. Actin served as a loading control. Representative data from two independent experiments are presented. (f) In vitro proliferation of Bcor ΔE4/y T-ALL cells. CD8 SP T-ALL cells from the spleen of T-ALL no. 4 were cultured on TSt-4 stromal cells in the presence of a γ-secretase inhibitor DAPT. Data are presented as the mean ± SEM of triplicate cultures. Representative data from two independent experiments are presented. ***, P < 0.001 by Student’s t test. (g) Quantitative RT-PCR analysis of Bcor and Bcl6 in various hematopoietic cell fractions. Hprt1 was used to normalize the amount of input RNA. Data are shown as the mean ± SD ( n = 3). Representative data from two independent experiments are presented. (h) Venn diagram of RefSeq genes up-regulated in DP thymocytes from Bcor ΔE4/y and Bcl6 Δ/Δ mice 4 wk after the injection of tamoxifen (more than twofold relative to the WT control). The numbers of genes in each group are indicated. The overlap between the two gene sets is statistically significant (P < 1 × 10 −242 ). (i) Snapshots of RNA sequence signals at the Myc gene locus in WT, Bcor ΔE4/y , and Bcl6 Δ/Δ DP thymocytes and Bcor ΔE4/y DP T-ALL cells. The structure of the Myc gene locus is indicated at the bottom. (j) In vitro proliferation of Bcor ΔE4/y and Bcl6 Δ/Δ thymocytes. DN1/2 thymocytes from Bcor ΔE4/y and Bcl6 Δ/Δ mice were cultured on TSt-4/DLL stromal cells in the presence of 10 ng/ml SCF, Flt3L, and IL-7. Data are presented as the mean ± SEM of triplicate cultures. (k) In vitro differentiation of Bcor ΔE4/y and Bcl6 Δ/Δ thymocytes. Culture conditions in j were switched to differentiation conditions by reducing the cytokine concentration to 2 ng/ml on day 14 of culture, and cells were cultured for a further 7 d. Differentiation was evaluated by flow cytometric analyses. Representative CD4 and CD8 expression profiles are depicted. The proportions of CD4 + CD8 + , CD4 + CD8 − , and CD4 − CD8 + thymocytes are as follows: WT, 25.8 ± 1.0, 21.7 ± 0.6, and 9.1 ± 0.1; Bcor ΔE4/y , 17.1 ± 0.6, 4.1 ± 0.1, and 24.0 ± 0.2; and Bcl6 Δ/Δ , 14.1 ± 0.8, 7.2 ± 0.2, and 16.3 ± 0.5, respectively ( n = 3). The proportions of CD4 SP and CD8 SP thymocytes are shown at right. **, P < 0.01; ***, P < 0.001 by Student’s t test. Representative data from three independent experiments are presented (j and k).

Article Snippet: Mixtures of solutions A and B were incubated at 95°C for 10 min. To detect histone proteins, cells were lysed in 2× SDS sample buffer, sonicated, and incubated at 95°C for 10 min. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and detected by Western blotting using the following antibodies: anti-BCOR , anti-BCL6 (sc-7388; Santa Cruz Biotechnology), anti-actin (clone C-4, SC-47778; Santa Cruz Biotechnology), anti-Flag (clone M2, F3165; Sigma-Aldrich), anti-HA (clone 3F10, 11867423001; Roche), anti–cleaved NOTCH1 (4147; Cell Signaling Technology), anti-PCGF1 (183499; Abcam), anti-RING1B (D139-3; MBL), anti-H2AK119ub (8240S; Cell Signaling Technology), and anti–histone H2A (ab18255; Abcam).

Techniques: Activation Assay, Quantitative RT-PCR, Sequencing, Mutagenesis, Variant Assay, Western Blot, Control, In Vitro, Cell Culture, Injection, Concentration Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Internal deletion of BCOR reveals a tumor suppressor function for BCOR in T lymphocyte malignancies

doi: 10.1084/jem.20170167

Figure Lengend Snippet: BCOR targets Myc and other NOTCH1 targets in DP thymocytes. (a) Distribution of ChIP-seq peaks of BCOR in thymocytes (q-value < 0.2) and NOTCH1 in T-ALL cells (data were retrieved from ; q-value < 10 −10 ) in the indicated regions (left and middle, respectively). Representative NOTCH1 target genes are indicated. Number of BCOR peaks with and without NOTCH1 peaks in close proximity (±1.0 kb) are depicted at right. (b) Snapshots of the ChIP-seq signals of BCOR and BCOR ΔE4/y at the Myc and Hes1 gene loci in WT and Bcor ΔE4/y thymocytes, respectively, and those of NOTCH1 in T-ALL cells. The structures of Myc and Hes1 gene loci including relevant exons are indicated at the bottom of each related snapshot. (c) Manual ChIP assays for Bcor at the Myc locus using an anti-BCOR antibody. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. Data are shown as the mean ± SD ( n = 3). *, P < 0.05 by Student’s t test; N.S., not significant. (d) Venn diagram of BCOR target genes in DP thymocytes and NOTCH1 target in T-ALL cells at the promoter region. The overlapping genes had BCOR and NOTCH1 peaks at their promoters in close proximity (≤1.0 kb). Representative NOTCH1 target genes are indicated. (e) Venn diagram of BCOR target genes at promoters and genes derepressed more than twofold in Bcor ΔE4/y DP thymocytes from WT DP thymocytes. Representative NOTCH1 target genes are indicated. (f) Venn diagram of the target genes of BCOR and BCOR ΔE4/y in WT and Bcor ΔE4/y thymocytes, respectively. Representative data from two independent experiments are presented (a–f).

Article Snippet: Mixtures of solutions A and B were incubated at 95°C for 10 min. To detect histone proteins, cells were lysed in 2× SDS sample buffer, sonicated, and incubated at 95°C for 10 min. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and detected by Western blotting using the following antibodies: anti-BCOR , anti-BCL6 (sc-7388; Santa Cruz Biotechnology), anti-actin (clone C-4, SC-47778; Santa Cruz Biotechnology), anti-Flag (clone M2, F3165; Sigma-Aldrich), anti-HA (clone 3F10, 11867423001; Roche), anti–cleaved NOTCH1 (4147; Cell Signaling Technology), anti-PCGF1 (183499; Abcam), anti-RING1B (D139-3; MBL), anti-H2AK119ub (8240S; Cell Signaling Technology), and anti–histone H2A (ab18255; Abcam).

Techniques: ChIP-sequencing, Immunoprecipitation

Journal: Blood

Article Title: RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2017-03-775536

Figure Lengend Snippet: RUNX1 is ubiquitously expressed in human T-ALL cells, and RUNX1 or CBFβ knockdown results in apoptosis. (A) Protein was isolated from human T-ALL cell lines and RUNX1, RUNX3, CBFβ, TAL1, MYB, NOTCH1, and MYC protein levels were determined by immunoblotting. Extracellular signal–regulated kinase 1/2 (ERK1/2) was used as a loading control. (B) The human T-ALL cell line Jurkat was infected with lentiviruses expressing a control shRNA or 2 shRNAs specific for RUNX1. RUNX1 mRNA and protein levels were examined by qRT-PCR and immunoblotting. (C) RUNX1 knockdown results in leukemic cell apoptosis. Control (GFP) and RUNX1 shRNA-transduced Jurkat cells were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry 6 days after infection. A representative flow profile is shown (left). The percentage of apoptotic cells was determined by Annexin V/7AAD staining and analyzed by flow cytometry. Four independent experiments were performed, and data are shown as means ± SD (right). (D) CBFβ knockdown also induces apoptosis. Control (GFP) or CBFβ shRNA-transduced Jurkat cells were stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry. Four independent experiments were performed, and data are shown as means ± SD (right). (E) CBFβ protein levels in control and knockdown cells were analyzed by immunoblotting. **P < .005; ***P < .0005; ****P < .0001, one-way ANOVA multiple comparisons test.

Article Snippet: To examine protein expression in human T-ALL cells, cells were lysed in modified radioimmunoprecipitation assay buffer, transferred to a membrane, and probed with antibodies to RUNX1 (ab23980, Abcam), RUNX3 (MAB3765, R&D Systems), TAL1 (sc-12984, Santa Cruz Biotechnology), MYB (05-175, EMD Millipore), NOTCH1 (Val1744, Cell Signaling Technology), MYC (N262, Santa Cruz Biotechnology), or extracellular signal-regulated kinase 1/2 (9102, Cell Signaling Technology).

Techniques: Knockdown, Isolation, Western Blot, Control, Infection, Expressing, shRNA, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Blood

Article Title: RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2017-03-775536

Figure Lengend Snippet: RUNX1 regulates a subset of TAL1- and NOTCH1-regulated genes. (A) mRNA was isolated from mouse T-ALL cells 48 hours after vehicle or 4-OHT treatment, and the expression of a subset of a RUNX1- and TAL1-regulated genes was determined by qRT-PCR. Three to 4 independent experiments were performed, and data are shown as means ± standard errors of the means (SEM). (B) Gene set enrichment analysis of RUNX1-regulated genes and genes changed on reactivation of NOTCH1 by γ-secretase inhibitor (GSI) washout (51). RUNX1 target genes that were significantly downregulated by RUNX1 knockdown in Jurkat cells were used as a data set (22). (C) The expression of a subset of NOTCH1-regulated genes in Runx1-deleted mouse T-ALL cells was determined by qRT-PCR. Three to 4 independent experiments were performed and data are shown as means ± SEM. *P < .05; **P < .005; ***P < .0005, Student t test.

Article Snippet: To examine protein expression in human T-ALL cells, cells were lysed in modified radioimmunoprecipitation assay buffer, transferred to a membrane, and probed with antibodies to RUNX1 (ab23980, Abcam), RUNX3 (MAB3765, R&D Systems), TAL1 (sc-12984, Santa Cruz Biotechnology), MYB (05-175, EMD Millipore), NOTCH1 (Val1744, Cell Signaling Technology), MYC (N262, Santa Cruz Biotechnology), or extracellular signal-regulated kinase 1/2 (9102, Cell Signaling Technology).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Knockdown

Journal: Blood

Article Title: RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2017-03-775536

Figure Lengend Snippet: RUNX1 is required for intracellular NOTCH1 binding and for chromatin accessibility at the N-Me. (A) H3K27ac, NOTCH1, and RUNX1 enrichment at the human MYC super-enhancer is shown in genome browser tracks (genome.ucsc.edu, human hg19). (B) The mouse genomic region (mm10) encompassing Myc and its enhancer loci located 1.27 Mb from the TSS are shown. The RBPJ- and RUNX-binding sites are depicted. (C) Recruitment of RUNX1, intracellular NOTCH1, H3K27ac, and H3 to the mouse Myc enhancer was determined by ChIP-qPCR in control or Runx1-deleted mouse T-ALLs. (D) The degree of open chromatin at the N-Me enhancer region in control or Runx1-deleted mouse T-ALLs was determined by ATAC-quantitative PCR. Data are the mean of 3 or 4 independent experiments and error bars represent ± SEM. *P < .05; **P < .005; ***P < .0005, two-way ANOVA multiple comparisons test.

Article Snippet: To examine protein expression in human T-ALL cells, cells were lysed in modified radioimmunoprecipitation assay buffer, transferred to a membrane, and probed with antibodies to RUNX1 (ab23980, Abcam), RUNX3 (MAB3765, R&D Systems), TAL1 (sc-12984, Santa Cruz Biotechnology), MYB (05-175, EMD Millipore), NOTCH1 (Val1744, Cell Signaling Technology), MYC (N262, Santa Cruz Biotechnology), or extracellular signal-regulated kinase 1/2 (9102, Cell Signaling Technology).

Techniques: Binding Assay, ChIP-qPCR, Control, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Soluble guanylate cyclase signalling mediates etoposide resistance in progressing small cell lung cancer

doi: 10.1038/s41467-021-26823-6

Figure Lengend Snippet: a Schematic of the working hypothesis. b Western blot for GUCY1B1 and synaptophysin (SYP) expression in SCLC cell lines ( n = 3). c HEY1 , GUCY1B1 and GUCY1A1 mRNA expression (RT-qPCR) of DMSO- or DAPT-treated H196 cells. n = 3, data are represented as mean ± SEM. P values from two-sided unpaired Student’s t- test. HEY1 p = 0.0001, GUCY1B1 p = 0.0002. d Western blot for GUCY1B1 and HES1 of DMSO- or DAPT-treated H196 cells. Quantification of GUCY1B1 (relative volume intensity, RVI) on the right, normalized to histone H3. n = 3, data are represented as mean ± SEM; p values from two-sided unpaired Student’s t- test. e Western blot for Notch1, HES1 and GUCY1B1 in sgNTA, sgNotch1-1 and sgNotch1-2 H1048 cells ( n = 3). f Western blot for N1ICD, HES1 and GUCY1B1 in empty vector control and N1ICD-overexpressing H1048 cells. Quantification of GUCY1B1 (RVI) on the right, normalized to H3. n = 3, data are represented as mean ± SEM. P values from two-sided unpaired Student’s t- test. N1ICD p = 0.0008, HES1 p = 0.0009. g Western blot for GUCY1B1 and SYP in CDX17 and CDX17P NE and Non-NE ex vivo cultures ( n = 2). h HEY1 , GUCY1B1 and GUCY1A1 mRNA expression (RT-qPCR) of DMSO- or DAPT-treated CDX17P Non-NE cells. n = 3, data are represented as mean ± SEM. P values from two-sided unpaired Student’s t- test. i HEY1 , GUCY1B1 and GUCY1A1 mRNA expression (RT-qPCR) of empty vector control or N1ICD-overexpressing CDX17P NE cells. n = 3, data are represented as mean ± SEM. P values from two-sided unpaired Student’s t- test. HEY1 p = 0.0005. j Notch1 ChIP-qPCR in H1048 cells overexpressing N1ICD. qPCR of RBPJ-binding sites in the GUCY1A1 and GUCY1B1 promoter. N1ICD binding to the HES1 promoter (positive control), binding to negative control region (negative control). n = 3, data are represented as mean ± SD. P values from two-sided unpaired Student’s t- test. See also Supplementary Fig. .

Article Snippet: ChIP was performed using the following antibodies: 5 μg Rabbit Notch1 antibody (Bethyl Laboratories, A301-895A, RRID:AB_1524102), 5 μg Rabbit (DA1E) mAb IgG XP Isotype Control (Cell Signaling Technology, 3900).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Ex Vivo, ChIP-qPCR, Binding Assay, Positive Control, Negative Control

Journal: Cancer discovery

Article Title: Gata3-controlled nucleosome eviction drives Myc enhancer activity in T-cell development and leukemia

doi: 10.1158/2159-8290.CD-19-0471

Figure Lengend Snippet: (A) Normalized 4C contact profiles in Jurkat cells (upper panel) and NOTCH1-induced mouse T-ALL cells (lower panel). Viewpoint is located in the MYC promoter (top tracks) or in N-Me (bottom tracks). 4C signal is merged across three replicates. The median, 20th and 80th percentiles of sliding 25Kb windows determine the main trend line. Color scale represents read coverage of sliding windows sized from 2 to 50Kb. (B) Analysis epigenetic of marks (yellow), epigenetic factor (gray) and transcription factor (blue) N-Me occupancy by ChIPseq in human T-ALL cells. Dotted lines mark the boundaries of N-Me. Scale bar is represented in the upper left corner. (C) Reverse ChIP identification of potential N-Me-binding factors. A N-Me DNA bait was incubated in the presence of nuclear extracts from Jurkat, ALL-SIL and HPB-ALL cells and recovered peptides were analyzed by mass spectrometry. The diagram represents the proteins recovered in one (purple), two (red) or all three (blue) cell lines analyzed. (D) N-Me evolutionary conservation tree. (E) Predicted ultraconserved transcription factor binding motifs in the N-Me sequence. PhyloP scores are shown above the sites.

Article Snippet: To analyze the binding of transcription factors to N-Me, we used wild type and GS1+2 Myc-rescued tumor cells and performed ChIP as describer before for Smarca4 using antibodies against cleaved Notch1 (Santa Cruz 6014-R, RRID: AB_650335), Ets1 (Santa Cruz 350, RRID: AB_2100688), Runx1 (Abcam ab23980, RRID: AB_2184205), Tcf1 (Santa Cruz 271453, RRID: AB_10649799), Tcf3/E2A (Santa Cruz 349X), and Tcf12/HEB (Cell Signaling 11825, RRID: AB_2797736).

Techniques: Binding Assay, Incubation, Mass Spectrometry, Sequencing

Journal: Cancer discovery

Article Title: Gata3-controlled nucleosome eviction drives Myc enhancer activity in T-cell development and leukemia

doi: 10.1158/2159-8290.CD-19-0471

Figure Lengend Snippet: (A) Quantification of the CD4+ CD8+ preleukemic cells in peripheral blood of mice transplanted with ΔE-NOTCH1-infected wild type (GS1+2 +/+), GS1+2 heterozygous (GS1+2 +/mut), and GS1+2 homozygous (GS1+2 mut/mut) mutant bone marrow progenitors. (B) Representative blood smear preparations in mice transplanted as in A eight weeks after transplant. (C) Kaplan-Meier survival curves (n=7) in mice transplanted as in A. (D) Survival analysis of mice transplanted with Rosa26TM-Cre NOTCH1-induced leukemias harboring a N-Me conditional and a N-Me GS1+2 mutant allele, and treated with vehicle only (N-Meflox/GS1+2mut) or tamoxifen (N-Me−/GS1+2mut) (n = 8 per group). (E) RT-qPCR analysis of Myc expression in vehicle treated (N-Meflox/GS1+2mut) and tamoxifen treated (N-Me−/GS1+2mut) NOTCH1-induced T-ALL N-Meflox/GS1+2mut tumor cells. (F) Growth curve of vehicle treated (N-Meflox/GS1+2mut) and tamoxifen treated (N-Me−/GS1+2mut) NOTCH1-induced T-ALL N-Meflox/GS1+2mut tumor cells. (G) Cell cycle analysis of NOTCH1-induced T-ALL N-Meflox/GS1+2mut tumor cells treated with vehicle (N-Meflox/GS1+2mut) or tamoxifen (N-Me−/GS1+2mut) for 3 days. (H) Analysis of apoptosis and cell death in NOTCH1-induced T-ALL N-Meflox/GS1+2mut tumor cells treated with vehicle (N-Meflox/GS1+2mut) or tamoxifen (N-Me−/GS1+2mut) for 3 days. The P value in A, E, F, G and H was calculated using two-tailed Student’s t-test. The P value in C and D was calculated using log-rank test.

Article Snippet: To analyze the binding of transcription factors to N-Me, we used wild type and GS1+2 Myc-rescued tumor cells and performed ChIP as describer before for Smarca4 using antibodies against cleaved Notch1 (Santa Cruz 6014-R, RRID: AB_650335), Ets1 (Santa Cruz 350, RRID: AB_2100688), Runx1 (Abcam ab23980, RRID: AB_2184205), Tcf1 (Santa Cruz 271453, RRID: AB_10649799), Tcf3/E2A (Santa Cruz 349X), and Tcf12/HEB (Cell Signaling 11825, RRID: AB_2797736).

Techniques: Infection, Mutagenesis, Quantitative RT-PCR, Expressing, Cell Cycle Assay, Two Tailed Test

Journal: Cancer discovery

Article Title: Gata3-controlled nucleosome eviction drives Myc enhancer activity in T-cell development and leukemia

doi: 10.1158/2159-8290.CD-19-0471

Figure Lengend Snippet: (A) ATAC-seq chromatin accessibility analysis of N-Me during T-cell differentiation. Dotted lines mark the boundaries of N-Me. Scale bar is represented in the upper left corner. (B) Heatmap representation of Myc expression in developing thymocytes. Myc RNAseq mRNA levels are color coded with red indicated higher levels and blue lower levels of expression. (C) ATAC-seq chromatin accessibility analysis of the N-Me enhancer in sorted DN3 thymocytes from 6-week old N-Me wild type and GS1+2 homozygous mutant mice (n=3). Normalized signal tracks for each genotype and differential chromatin accessibility heatmap are shown. (D) Nucleosome occupancy profiles as in C. Black bars indicate nucleosome-free regions. (E) Smarca4 occupancy of N-Me in DN3 thymocytes from GS1+2 heterozygous mutant mice. Expected sequences for the wild type and mutant GATA site alleles are indicated below the chromatograms. (F)Transcription factor occupancy of N-Me in wild type and Myc-rescued GS1+2 mutant NOTCH1-induced T-ALL lymphoblasts. PCR signal is normalized to input chromatin and to the average signal in wild type tumors. Error bars represent standard deviation between technical replicates. (G) ATAC-seq chromatin accessibility analysis of the N-Me enhancer in mouse DP thymocytes and in DP T-ALL lymphoblast cells. Dotted lines mark the boundaries of N-Me. Scale bar is represented in the upper right corner. (H) ATAC-seq chromatin accessibility analysis of the N-Me enhancer in human DP thymocytes and in two independent DP T-ALL samples as in G.

Article Snippet: To analyze the binding of transcription factors to N-Me, we used wild type and GS1+2 Myc-rescued tumor cells and performed ChIP as describer before for Smarca4 using antibodies against cleaved Notch1 (Santa Cruz 6014-R, RRID: AB_650335), Ets1 (Santa Cruz 350, RRID: AB_2100688), Runx1 (Abcam ab23980, RRID: AB_2184205), Tcf1 (Santa Cruz 271453, RRID: AB_10649799), Tcf3/E2A (Santa Cruz 349X), and Tcf12/HEB (Cell Signaling 11825, RRID: AB_2797736).

Techniques: Cell Differentiation, Expressing, Mutagenesis, Standard Deviation

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Heatmap of known Notch1 target genes and glycolytic genes identified by RNA-seq using A549 cells stably transfected with Notch1 short hairpin RNA (shRNA) or control shRNA. Western blot shows the knockdown of Notch1 expression. b KEGG pathway analysis of genes differentially expressed between A549 cells stably transfected with Notch1 shRNA or control shRNA. c , d The mRNA and protein expression of glycolytic genes in A549 cells stably transfected with Notch1 shRNA or control shRNA were examined by qRT-PCR ( c ) and western blot ( d ) respectively. e ChIP analysis of Notch1 occupancy on promoters of glycolytic genes in A549 cells. IgG: normal serum. The different number after each gene represents the regions containing different Notch1-binding sites. The graph shows the percentage of input. f , g A549 cells were transfected with empty vector (EV), Notch1 intracellular domain (ICD), or TAZ shRNA. Glucose uptake, pyruvate level, lactate production level ( f ), and extracellular acidification rate (ECAR) ( g ) were examined. * P < 0.05.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: RNA Sequencing, Stable Transfection, Transfection, shRNA, Control, Western Blot, Knockdown, Expressing, Quantitative RT-PCR, Binding Assay, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Cellular extracts from A549 cells stably expressing FLAG (control) or FLAG-Notch1 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptide. The eluates were resolved by SDS-PAGE and silver stained. The differential protein bands were retrieved and analyzed by mass spectrometry. b A549 cells were transfected with Notch1 ICD, p300 shRNA, pCAF shRNA, or empty vector (EV). Glycolytic gene expression was measured using qRT-PCR. * P < 0.05. c ChIP analysis of Notch1, p300, and pCAF occupancy on glycolytic gene promoters in A549 cells. The graph shows the percentage of input. d Re-ChIP analysis of the occupancy of Notch1 and p300 or pCAF on the glycolytic gene promoters in A549 cells. e A549 cells were immunoprecipitated with anti-p300, anti-pCAF, or normal IgG, and the precipitates were analyzed by immunoblot with the indicated antibodies. IP immunoprecipitation. f A549 cells stably transfected with Notch1 were co-transfected p300 shRNA or pCAF shRNA. The protein expression of glycolytic genes was examined using western blot assay. g Notch1, p300, pCAF, and histone H3 and H4 acetylation occupancy on the promoters of indicated glycolytic genes in A549 cells transfected with Notch1 shRNA, p300 shRNA or pCAF shRNA was examined using ChIP assay.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Stable Transfection, Expressing, Control, SDS Page, Staining, Mass Spectrometry, Transfection, shRNA, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a The reporter activity of Notch1 and Hes1 in A549 cells transfected with TAZ was measured by luciferase reporter assay. * P < 0.05. Relative luciferase activity was performed to identify Notch responsive region in the Hes1 promoter. b The protein levels of Notch1 and Hes1 induced by TAZ overexpression in A549 cells were examined by western blot assay. c Notch1 ICD associated with endogenous TAZ in A549 cells. Immunoprecipitation (IP) was performed using antibodies of Notch1 ICD and TAZ, and coprecipitated protein was analyzed by western blot assay. d Comparison of Notch1 ChIP-seq signal (expressed as normalized read density, RPKM) in active enhancers with or without TAZ peaks in A549 cells treated with DMSO, Brontictuzumab (2 μm for 5 h), or cells transfected with TAZ shRNA. * P < 0.05. e TAZ binding at enhanc e rs of TAZ target genes in TAZ 4SA overexpressing A549 cells by ChIP-qPCR analysis. DNA enrichment was calculated and presented as fold vs control cells. f ChIP-qPCR analysis showed Notch1 binding on enhancers and promoters (TSS, transcription start site) of TAZ targets upon TAZ 4SA overexpressing in A549 cells, but not in the presence of Brontictuzumab (2 μm for 5 h).

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Over Expression, Western Blot, Immunoprecipitation, Comparison, ChIP-sequencing, shRNA, Binding Assay, ChIP-qPCR, Control

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a Protein analysis of Jagged1, TAZ, TEAD1, and Hes1 in A549 cell lysates after transfected with TAZ and concomitant silencing of TEAD1 by shRNA. b Hes1 reporter assay in the presence (+) or absence (−) of Notch1 ICD, or TAZ, N = 3. Results of luciferase reporter assays are shown. * P < 0.05. c TEAD1-reporter luciferase assay in the presence (+) or absence (−) of TAZ or nTEAD1, N = 3. d Jagged1 luciferase reporter assay in the presence (+) or absence (−) of Notch1 ICD, TAZ, or Mst1, N = 3. * P < 0.05. Cells were transfected with or without MST1, phosphorylated TAZ and TAZ were analyzed by western blot assay. e ChIP assay for Notch1 or TAZ in A549 cells transfected with Notch1, TAZ, and plasmid of either Jagged1-ECR1 or ECR6. Data are presented as fold enrichment over an IgG ChIP performed with the same samples. f ChIP assay for TAZ in A549 cells at Jagged1-ECR1, ECR6, and Hes1 promoter. N = 3. * P < 0.05.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Transfection, shRNA, Reporter Assay, Luciferase, Western Blot, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: a The proliferation curve of A549 cells transfected with Notch1, TAZ, TAZ shRNA or empty vector. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. b The proliferation curve of A549 cells transfected with Notch1 or empty vector, treated with 2.5 mM 2-DG as indicated. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. c The proliferation curve of A549 cells transfected with TAZ or empty vector, treated with 2.5 mM 2-DG as indicated. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. d The proliferation curve of A549 cells transfected with Notch1, TAZ or empty vector, treated with 0.1 mM Oligomycin in normal culture medium (containing 25 mM glucose) as indicated. Cell proliferation was determined by the CCK-8 Kit. * P < 0.05. e MicroPET-CT imaging of nude mice to determine FDG uptake in mice with subcutaneous xenograft lung cancer model established with A549 cells transfected with empty vector, p300 shRNA, pCAF shRNA, Notch1, TAZ, Notch1 shRNA, or TAZ shRNA. Representative 18F FDG microPET images are shown with arrowheads indicating xenografted lung cancers at treatment end (day 35). Quantification of 18F FDG uptake in tumors is shown as %IDmean/g. * P < 0.05. f Xenograft tumors were established as in ( b ) and the growth curve was plotted. * P < 0.05. g A549 cells stably expressing Notch1, TAZ, or LDHA shRNA were subcutaneously injected into nude mice. 2-DG was used as indicated. The growth curve was plotted. * P < 0.05. h Representative expression of Notch1 and TAZ by immunohistochemistry assay of 23 lung cancer patients. The correlation of glucose uptake with Notch1 or TAZ expression was determined using the Mann–Whitney U test. Scale bar = 50 μm. Original magnification: ×100. * P < 0.05.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Transfection, shRNA, Plasmid Preparation, CCK-8 Assay, Imaging, Stable Transfection, Expressing, Injection, Immunohistochemistry, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer

doi: 10.1038/s41419-021-04124-6

Figure Lengend Snippet: Notch1 forms a positive feedback loop with TAZ and promotes glycolytic gene expressions through interaction with p300 and pCAF. Increased levels of extracellular lactate via Notch1/TAZ loop inhibits cytotoxic T-cell activity, which contributes to lung cancer invasion.

Article Snippet: The Notch1 promoter construct (−2001/−1) was generated from human genomic DNA corresponding to the sequence from −2001 to −1 (relative to the transcriptional start site) and cloned to the pRL-TK-Basic vector (Promega, Madison, WI, USA).

Techniques: Activity Assay

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: NOTCH1 expression is up-regulated in cardiac macrophages after lipopolysaccharide (LPS) treatment. A - B , representatively echocardiographic M-model figures and analysis of heart function from LPS and PBS group ( n = 8 for PBS, n = 15 for LPS). C , survival rate of mice records for a 72-hour period from LPS and PBS group ( n = 10 for PBS, n = 15 for LPS). D , the representative immunofluorescence (IF) CD68 (green) and NOTCH1 (red) in the hearts of PBS or LPS-injected mice. Yellow indicates colocalization of NOTCH1 in macrophages. Scale bar, 100 μm. E , RT-qPCR analysis of Notch1, notch2, DLL4 and Hes1 mRNA expression in bone marrow-derived macrophages from LPS and PBS group ( n = 10 for PBS, n = 15 for LPS). F , the equation of regression between NOTCH1 expression with left ventricular ejection fraction (EF) or fractional shortening (FS) from LPS group. G , immunoblot analysis of NOTCH1 protein expression in bone marrow-derived macrophages from LPS and PBS group ( n = 7). *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Expressing, Immunofluorescence, Injection, Quantitative RT-PCR, Derivative Assay, Western Blot

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: Macrophage specific-knockout NOTCH1 declines cardiac injury post LPS challenge. A - B , representatively echocardiographic M-model figures and analysis of heart function in EF, FS, LV internal volumes at end-systole and end-diastole (LVESV and LVEDV) from wild type (WT) and macrophage specific-knockout NOTCH1 mice post LPS challenge ( n = 6 for PBS, n = 15 for LPS). C , measurements of survival rate in WT and macrophage specific-knockout NOTCH1 mice post LPS challenge ( n = 8 for PBS, n = 10 for LPS). D , the representative TUNEL staining and analysis of apoptotic cardiomyocyte rate from WT and macrophage specific-knockout NOTCH1 mice post LPS challenge ( n = 5). Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Knock-Out, TUNEL Assay, Staining

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: Overexpression of NOTCH1 aggravates cardiac injury post LPS challenge. A - B , representatively echocardiographic M-model figures and analysis of heart function from AAV-NC and AAV-F4/80-NICD mice post LPS challenge ( n = 6). C , measurements of survival rate in AAV-NC and AAV-F4/80-NICD mice post LPS challenge ( n = 6 for PBS, n = 8 for LPS). D , the representative TUNEL staining and analysis of apoptotic cardiomyocyte rate from AAV-NC and AAV-F4/80-NICD mice post LPS challenge (n = X). Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Over Expression, TUNEL Assay, Staining

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: NOTCH1 ΔMyelo decreases mitochondrial damage after LPS + adenosine triphosphate (ATP) treatment. A , flow cytometry (FACS) analysis of mitoROS ratio in NOTCH1 ΔMyelo or WT group after LPS + ATP treatment ( n = 5). B , analysis of mitochondrial membrane potential in NOTCH1 ΔMyelo or WT group after LPS + ATP treatment ( n = 4). C , FACS analysis of damage mitochondria in NOTCH1 ΔMyelo or WT group after LPS + ATP treatment ( n = 4). D , FACS analysis of apoptotic cell rate in NOTCH1 ΔMyelo or WT group after LPS + ATP treatment ( n = 5). E , ELISA analysis of IL-1β and TNF-α secretion level in NOTCH1 ΔMyelo or WT group after LPS + ATP treatment combining with mito-TEMPO ( n = 5). n.s. indicates nonsignificant. ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Flow Cytometry, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: NOTCH1 ΔMyelo down regulates NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after LPS + ATP treatment. A , Immunoblot and quantification analysis of GSDMD, cleaved caspase-1, Caspase 11 and NLRP3 in NOTCH1 knockout macrophages after LPS + ATP treatment. B , ELISA analysis of IL-1β, IL-18, and TNF-α secretion level in NOTCH1 ΔMyelo or WT group after LPS + ATP treatment during 12 h ( n = 4). C , ELISA analysis of IL-1β, IL-18, and TNF-α secretion level in NOTCH1 ΔMyelo or WT group after LPS, ATP and LPS + ATP treatment ( n = 4). D , ELISA analysis of IL-1β, IL-18, and TNF-α secretion level in NOTCH1 ΔMyelo or WT group after LPS + nigericin treatment ( n = 4). n.s. indicates nonsignificant. ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Activation Assay, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: NOTCH1 ΔMyelo promotes mitophagy after LPS + ATP treatment. A - C , the representative IF staining and analysis of mitophagy flux in NOTCH1 knockout macropahges after LPS + ATP treatment (n = 5). Scale bar, 20 µm. D , the representative WB and analysis of mitophagy and autophagy-relative protein expression level in NOTCH1 knockout macrophages after LPS + ATP treatment (n = 3). E , transmission electron microscopy of morphological changes in mitochondria (arrow, mitochondria within autophagosomes; red‘N’, nucleus). Scale bar, 2 μm. ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Staining, Knock-Out, Expressing, Transmission Assay, Electron Microscopy

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: Suppressing mitophagy abolishes cardiac protection of NOTCH1 knockout post LPS challenge. A - B , representatively echocardiographic M-model figures and analysis of heart function from WT and NOTCH1 ΔMyelo mice after mdivi treatment ( n = 6 for WT, n = 7 f CH1 ΔMyelo ). C , the representative TUNEL staining and analysis of apoptotic cardiomyocyte rate from WT and NOTCH1 ΔMyelo mice after mdivi treatment ( n = 5). Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Knock-Out, TUNEL Assay, Staining

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: NOTCH1 up-regulates Mst1 expression via binding CSL elements. A , integrative Genomics Viewer genome browser tracks show the level of NICD1 enrichment near the Mst1 transcription start site in ChIP samples (red) over input (blue). The transcription start site is shown by the dashed line. B , the schematic structure of NICD binding elements in promoter of murine MST1 gene. C - E , ChIP-qPCR on these sites in cultured BMDMs after LPS + ATP treatmen. ( n = 3). F , ChIP-qPCR for NICD and CSL in cultured Raw264.7 cells after LPS + ATP treatment. ( n = 3). G , the schematic structure of designed sequences in MST1 promoter for luciferase assay. H , Luciferase assay in cultured Raw264.7 cells treats with vehicle, DAPT after LPS + ATP treatment. ( n = 3). I , luciferase assay in cultured Raw264.7 cells treats with scramble and shRNAs of NICD after LPS + ATP treatment. ( n = 3). J , luciferase assay in cultured BMDMs overexpressing 3xflag-GFP or 3xflag-NICD1 after LPS + ATP treatment. ( n = 3). K - L , effects of HRE, NF-κB, or CSL site mutation on MST1 promoter activity in cultured BMDMs treats with DAPT, LPS + ATP, LPS + ATP + DAPT. ( n = 3). M - N , the representative IF staining and analysis of mitophagy flux in Mst1 overexpression macrophages after LPS + ATP treatment ( n = 3). Scale bar, 20 μm. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Expressing, Binding Assay, ChIP-qPCR, Cell Culture, Luciferase, Mutagenesis, Activity Assay, Staining, Over Expression

Journal: Biology Direct

Article Title: Macrophage Notch1 drives septic cardiac dysfunction by impairing mitophagy and promoting NLRP3 activation

doi: 10.1186/s13062-025-00657-4

Figure Lengend Snippet: A schematic illustration showing that NOTCH1 in macrophages drives septic cardiac dysfunction by inhibiting mitophagy and inducing NLRP3 activation

Article Snippet: Notch1 flox/flox (Notch f/f ) and LysM-Cre mouse strains (both on a C57BL/6 background) were obtained from GemPharmatech Co. Ltd. (Nanjing, China).

Techniques: Activation Assay

Journal: Blood cancer discovery

Article Title: A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia

doi: 10.1158/2643-3230.BCD-20-0201

Figure Lengend Snippet: Identification of PE, a PTEN enhancer in T-ALL. A, H3K27ac Hi-ChIP, 4C-seq, ChIP-seq, GRO-seq and ATAC-seq tracks in human T-ALL cells. Top track shows H3K27ac Hi-ChIP interactions with the PTEN promoter in CUTTl1 T-ALL cells at FDR <1E-15. Upper tracks show 4C-seq data in DND41 (blue), HPB- ALL (red) or JURKAT (green) T-ALL cells, using either the PTEN promoter or the PE enhancer as the viewpoints. 4C signal is merged across three independent replicates per condition. Middle tracks show ChIP-seq analyses in different T-ALL cell lines for the presence of epigenetic marks or enhancer-associated factors (orange). CTCF motifs are indicated by arrows (red arrow: forward core motif, blue arrow: reverse core motif). Lower tracks show GRO-seq data from CUTTL1 cells (pink). Bottom track shows the PTEN TAD (hg19). The PTEN promoter and the PE enhancer are highlighted by orange columns. B, Analysis of epigenetic marks (yellow), epigenetic factos (gray) and transcription factor (blue) PE occupancy by ChIP-seq in human T-ALL cells. PE enhancer is highlighted by an orange column. C, H3K27ac mark by ChIPmentation around the PE enhancer (highlighted in orange) in 6 independent human primary T-ALLs. D, ATAC-seq profile around the PE enhancer (highlighted in orange) in 3 independent human primary T-ALLs (GSE124223). E, 4C-seq, ChIP-seq and ATAC-seq tracks in mouse T-ALL cells. Upper tracks show 4C-seq data from NOTCH1-induced mouse primary T-ALLs driven by either a NOTCH1-HDΔP construct (brown) or a NOTCH1-ΔE construct (green), using either the Pten promoter or the PE enhancer as the viewpoints. 4C signal is merged across three independent replicates per condition. Middle tracks show ChIP-seq (orange) of H3K27ac mark in mouse T-ALL cells and CTCF binding in mouse Th1 cells. CTCF motifs are indicated by arrows (red arrow: forward core motif, blue arrow: reverse core motif). Lower tracks show ATAC-seq data from a mouse primary T-ALL (blue), as well as the track showing the Pten TAD (mm10). The Pten promoter and the PE enhancer are highlighted by orange columns.

Article Snippet: We performed 4C-seq analysis in JURKAT, HPB-ALL and DND41 human T-ALL cells and HDΔP-NOTCH1-induced or ΔE-NOTCH1-induced mouse primary T-ALL lymphoblasts (previously described( 11 )) following published protocols ( 69 ) and using the restriction enzymes HindIII (R3104M, NEB) and DpnII (R0543M, NEB).

Techniques: HiChIP, ChIP-sequencing, Construct, Binding Assay

Journal: Blood cancer discovery

Article Title: A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia

doi: 10.1158/2643-3230.BCD-20-0201

Figure Lengend Snippet: Secondary loss of PE leads to accelerated NOTCH1-induced T-ALL progression and reduced levels of PTEN in mouse and human T-ALL. A, Schematic of retroviral-transduction protocol or the generation and analysis of PE conditional knockout NOTCH1-induced T-ALL. B, Kaplan-Meier curves of mice transplanted with PE conditional knockout ΔE-NOTCH1-induced T-ALL and treated in vivo with vehicle (control) or tamoxifen, to induce isogenic deletion of PE. ***P ≤ 0.005 values calculated using the log-rank test. C, Quantitative RT-PCR analysis of Pten expression in tumor cells isolated from PE conditional knockout leukemia–bearing mice treated with vehicle only (n = 7) or tamoxifen (n = 8) in vivo. Graph show the mean values, and the error bars represent the s.d. ***P ≤ 0.005 was calculated using two-tailed Student’s t test. D, Western blot analysis of PTEN expression in tumor cells isolated from PE conditional knockout leukemia–bearing mice treated with vehicle only (n =3) or tamoxifen (n = 3) in vivo. E, Heat map representation of the top 81 differentially expressed genes between control and tamoxifen-treated PE conditional knockout NOTCH1-induced leukemias. Cutoffs used: Wald statistic < −5 or > 5; P-adjusted value < 1E-04; sorted based on mean expression levels in tamoxifen-treated samples (for full list of significantly downregulated genes upon tamoxifen treatment, see Supplementary Fig. S12A). The scale bar shows color-coded differential expression with red indicating higher levels of expression and blue indicating lower levels of expression. F, Gene Set Enrichment Analysis (GSEA) of genes regulated by PTEN in vehicle only-treated compared to tamoxifen-treated PE conditional knockout NOTCH1-induced leukemia cells in vivo. G, H3K27ac ChIP-seq mark in DND41 T-ALL cells along the PTEN-containing TAD and schematic representation of chromosome 10q23 focal deletions (red bars) found in human T-ALL. H, PTEN mRNA expression levels in human primary T-ALL samples (n=360). Samples are subdivided according to the presence/absence of PTEN coding sequence (CDS) deletions, the presence/absence of PTEN CDS deletions affecting it transcriptional start site (TSS) and the presence/absence of PE focal deletions. P value was calculated using t-test. I, PE focal deletions found specifically in T-ALL but not B-ALL. P value calculated using Fisher’s exact test.

Article Snippet: We performed 4C-seq analysis in JURKAT, HPB-ALL and DND41 human T-ALL cells and HDΔP-NOTCH1-induced or ΔE-NOTCH1-induced mouse primary T-ALL lymphoblasts (previously described( 11 )) following published protocols ( 69 ) and using the restriction enzymes HindIII (R3104M, NEB) and DpnII (R0543M, NEB).

Techniques: Transduction, Knock-Out, In Vivo, Quantitative RT-PCR, Expressing, Isolation, Two Tailed Test, Western Blot, ChIP-sequencing, Sequencing

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: SIRT1 is overexpressed in T-ALL downstream of a NOTCH1-bound enhancer. A, Box-plot showing SIRT1 expression among T-ALL samples (n=57) and physiological thymocyte subsets (n=21) . Quantile normalization was performed across samples. Boxes represent first and third quartiles and the line represents the median. Whiskers represent the upper and lower limits ( P <0.001 using Mann-Whitney U-Test; FDR<0.05 using Benjamini-Hochberg correction). B, Western blot analysis of SIRT1 and ACTIN expression in human peripheral blood mononuclear cells (PBMNC), CD4+ T-cells or normal human thymocytes, as compared to human T-ALL cell lines. C, GSI washout experiments in CUTLL1 T-ALL cells, treated with GSI (Compound E, 1μM) for 3 days, washed twice, and incubated 4h in the presence or absence of 20μM cycloheximide . To control for GSI “off-NOTCH” effects, cells were also transduced with a dominant-negative MAML1 (DN-MAML1). (n=3 per condition; *** P < 0.005 using two-tailed Student t -test; NS, not significant). D, Western blot analysis of NOTCH1 (ICN1), SIRT1 and ACTIN expression in triplicates from DND41 or HPB-ALL human T-ALL cells treated with DBZ (250nM) for 3 days or mouse T-ALL cells treated with DBZ (250nM) for 24h. E, Epigenetic profiling around the SIRT1 promoter in human T-ALL showing ChIP-seq tracks in human T-ALL cell lines and ATAC-seq tracks in human T-ALL primary samples. N-Se enhancer highlighted in orange. F, Luciferase reporter activity in JURKAT cells of a pGL4 promoter empty construct (pGL4-Luc), a pGL4 promoter plus the human N-Se enhancer in the forward (NSe(+)-Luc) or reverse (NSe(-)-Luc) orientation. Data from three independent electroporation replicates are shown. *** P < 0.005 using two-tailed Student t -test. G, Genotyping of JURKAT single-cell clones harboring a N-Se homozygous deletion. JURKAT cells not electroporated (WT) are shown as controls. H, SIRT1 protein expression levels via western blot analysis in JURKAT control cells or four independent JURKAT single-cell clones with N-Se homozygous deletion. I, SIRT1 protein expression levels via western blot analysis in DND41 cells harboring either a dCas9-VP64 or dCas9-KRAB construct, and infected with gRNAs targeting either the SIRT1 promoter transcriptional start site (TSS) or two independent gRNAs targeting N-Se.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Expressing, MANN-WHITNEY, Western Blot, Incubation, Transduction, Dominant Negative Mutation, Two Tailed Test, ChIP-sequencing, Luciferase, Activity Assay, Construct, Electroporation, Clone Assay, Infection

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: SIRT1 inhibition shows antileukemic and synergistic effects with NOTCH1 inhibition. A-B, SIRT1 mRNA expression levels ( A ) and protein expression levels ( B ) in DND41 cells harboring two independent doxycycline-inducible shRNAs targeting SIRT1 with concomitant GFP expression or a non-targeting shRNA control, 3 days after doxycycline induction. C, Proliferation curve (left) and cell quantification at day 9 (right) of DND41 cells upon Doxycycline-induced expression of a control shRNA or shRNAs targeting SIRT1 . D-E , Representative flow cytometry plots from triplicate samples of annexin V (apoptotic cells) and 7-AAD (dead cells) staining ( D ) and quantification of apoptosis ( E ) of DND41 cells 9 days after Doxycycline-induced expression of a control shRNA or shRNAs targeting SIRT1 . Numbers in quadrants indicate percentage of cells. F, Proliferation curve (left) and cell quantification at day 9 (right) of DND41 cells treated with vehicle (DMSO), EX-527 (90μM), DBZ (250nM), or EX-527 and DBZ in combination. G , Isobologram analysis of DBZ and EX-527 treatment after 6 days in DND41 cells. The value for the combination index at ED50 is marked in blue. The ED50 for each drug is marked in black. H-I , Representative flow cytometry plots from triplicate samples of annexin V (apoptotic cells) and 7-AAD (dead cells) staining ( H ) and quantification of apoptosis ( I ) of DND41 cells treated with vehicle (DMSO), EX-527(90μM), DBZ (250nM) or EX-527 and DBZ in combination at Day 9. Numbers in quadrants indicate percentage of cells. * P < 0.05 and *** P < 0.005 in Figs. , and 1F using one-way analysis of variance (ANOVA). * P < 0.05 ** P < 0.01 and *** P < 0.005 in Figs. and using two-way ANOVA for multiple comparisons.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, Expressing, shRNA, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: SIRT1 promotes T-ALL development and confers resistance to NOTCH1 inhibition in vivo . A, Schematic of retroviral-transduction protocol for the generation of NOTCH1-induced T-ALLs from Sirt1 -overexpressing ( Sirt1 TG ) or wild-type control littermate ( Sirt1 WT ) mice. B, Kaplan-Meier curves of mice transplanted with E-NOTCH1 infected Sirt1 WT and Sirt1 TG hematopoietic progenitors (n=10 per genotype). * P < 0.05 value was calculated using the log-rank test. C, Western blot analysis of SIRT1 and ACTIN expression in leukemic spleens from terminally ill mice from survival curve in B. D, Schematic of retroviral-transduction protocol for the generation of NOTCH1-induced T-ALLs from inducible Sirt1 -conditional knockout mice. Two days upon transplantation of NOTCH1-infected Sirt1 flox/flox -Rosa26 Cre-ERT2/+ progenitors, mice were treated with corn oil vehicle ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ), in order to induce isogenic loss of Sirt1 . E, Kaplan-Meier curves of mice transplanted with NOTCH1-infected Sirt1 flox/flox -Rosa26 Cre-ERT2/+ progenitors (n=10 and treated with vehicle ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) as in D (n=10 per genotype). * P < 0.05 value was calculated using the log-rank test. F, Western blot analysis of SIRT1 and ACTIN expression in leukemic spleens from terminally ill mice from survival curve in E. G, Schematic for transduction of either wild-type Sirt1 or a deacetylase-dead H355A Sirt1 mutant concomitantly expressing the mCherry fluorescent protein in NOTCH1-induced GFP+ primary T-ALL cells followed by transplantation into mice, which were subsequently treated daily with DBZ in vivo . H, Peripheral blood leukemia infiltration in mice harboring NOTCH1-induced T-ALL cells expressing mCherry and Sirt1 wild-type or H355A-mutant upon continuous daily treatment with DBZ. Changes in leukemia cell counts of noninfected (mCherry-negative) cells are shown as an internal control. Error bars, median ± s.d.; P values were calculated using two-tailed Student t -test (n=5 mice per group); NS, not significant. I, C, Western blot analysis of SIRT1 and GAPDH expression in leukemic spleens from non-DBZ-treated, terminally ill mice from G.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, In Vivo, Transduction, Infection, Western Blot, Expressing, Knock-Out, Transplantation Assay, Histone Deacetylase Assay, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: Secondary loss of SIRT1 in established leukemias leads to antileukemic and synergistic effects with NOTCH1 inhibition in vivo . A, Schematic of retroviral-transduction protocol for the generation of NOTCH1-induced T-ALLs from inducible Sirt1 -conditional knockout mice, followed by transplant into secondary recipients treated with vehicle ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) and vehicle or DBZ. B, Kaplan-Meier survival curves of mice harboring Sirt1 -positive and Sirt1 -deleted isogenic leukemias treated with 4 cycles of vehicle or DBZ (5 mg/kg) on a 4-days-ON (red blocks) and 3-days-OFF schedule (log-rank test; ** P < 0.01; *** P < 0.005; n = 10 per group). C, Western blot analysis of SIRT1 and ACTIN expression in leukemic spleens from terminally ill mice from survival curve in B. D-E , Quantitative RT-PCR analysis of Sirt1 mRNA expression (D) and western blot analysis of SIRT1 protein levels (E) in tumor cells isolated from E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . F-G, Tumor burden in E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo as revealed by total spleen weight (F) and total spleen cell numbers (G). H-I , Representative flow cytometry plots from of annexin V (apoptotic cells) and 7-AAD (dead cells) staining ( H ) and quantification of apoptosis ( I ) in leukemic spleens from E-NOTCH1-induced Sirt1 conditional knockout leukemia– bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . (n = 5 per treatment; ** P < 0.01 and *** P < 0.005 in using two-tailed Student t -test).

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, In Vivo, Transduction, Knock-Out, Western Blot, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: Metabolic consequences of secondary loss of SIRT1 in established leukemias in vivo . A, Western blot analysis of AMPK protein levels and activation, 4E-BP1 levels and activation, and ATF4 levels in tumor cells isolated from E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . B, Significantly altered metabolites (upregulated in red, downregulated in blue) upon tamoxifen-induced isogenic loss of Sirt1 in leukemic spleens from mice treated as in A, ranked by P value (–log10 transformed). C-D, Relative abundance of indicated glycolytic intermediates (C) or glutamine/aspartate-related metabolites (D) upon tamoxifen-induced isogenic loss of Sirt1 in leukemic spleens from mice treated as in A. (n = 5 per treatment; * P < 0.05, ** P < 0.01 and *** P < 0.005 in Figs. 5A-D using two-tailed Student t -test). E, Oxygen consumption rate (OCR) in response to the indicated mitochondrial inhibitors in a E-NOTCH1-induced Sirt1 conditional knockout leukemia-derived cell line under basal conditions or 2-days after 4-Hydroxytamoxifen-induced isogenic loss of Sirt1 , measured in real time using a Seahorse XF24 instrument. Data are presented as +/- SD of n = 5 wells.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: In Vivo, Western Blot, Activation Assay, Isolation, Knock-Out, Transformation Assay, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: Secondary loss of SIRT1 leads to hyperacetylation of KAT7 and a transcriptional signature driven by KAT7 inhibition. A, Schematic representation of acetyl-proteomic experiments in tumor cells isolated from E-NOTCH1 or HD P-NOTCH1- induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . Venn diagram shows significantly hyperacetylated targets consistently found upon SIRT1 loss in both leukemias. B, Heatmap representation of the top differentially expressed genes between control ( Sirt1 +/+ ) and tamoxifen-treated ( Sirt1 -/- ) Sirt1 conditional knockout E-NOTCH1–induced leukemias. Cutoffs used: Wald statistic < −8 or > 8; P-adjusted value < 0.005; sorted based on mean expression levels. Scale bar shows color-coded differential expression, with red indicating higher levels of expression and blue indicating lower levels of expression. C, GSEA of genes regulated by KAT7 in vehicle only–treated ( Sirt1 +/+ ) compared with tamoxifen–treated ( Sirt1 -/- ) E-NOTCH1-induced Sirt1 conditional knockout leukemia cells in vivo . D, Western blot analysis (left) and quantification (right) of H4 total protein levels and H4K12ac levels in tumor cells isolated from E-NOTCH1- induced Sirt1 conditional knockout leukemia–bearing mice 2 days after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . ** P < 0.01 using two-tailed Student t -test. E, Western blot analysis (left) and quantification (right) of H4 total protein levels and H4K12ac levels in DND41 cells harboring two independent doxycycline-inducible shRNAs targeting SIRT1 with concomitant GFP expression or a non-targeting shRNA control, 3 days after doxycycline induction. F, Bar graph showing the number of significantly downregulated (blue) or upregulated (red) H4K12ac-containing genomic regions from H4K12ac ChIP-seq analyses in tumor cells isolated from E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . G, Volcano plot showing H4K12ac downregulated region genes (shrunken LFC < -0.3) in blue (n=2319) and the rest of the regions in light grey. Horizontal dashed line corresponds to the adjusted P value threshold of 0.001. Vertical dashed lines correspond to log 2 FC changes of +0.25 and -0.25. 303/2319 were significantly downregulated at the gene expression level (hypergeometric test P value = 9.48E-40).

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, Isolation, Knock-Out, In Vivo, Expressing, Western Blot, Two Tailed Test, shRNA, ChIP-sequencing

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: KAT7 partially mediates the antileukemic effects of loss of SIRT1. A, Relative cell proliferation of a E-NOTCH1-induced Sirt1 conditional knockout leukemia-derived cell line upon treatment with ethanol ( Sirt1 +/+ ) or 4-Hydroxytamoxifen ( Sirt1 -/- ) and different concentrations of the KAT7 inhibitor WM-3835 in vitro . * P < 0.05 and *** P < 0.005 using two-tailed Student t -test. B, Western blot analysis (left) and quantification (right) of a E-NOTCH1-induced Sirt1 conditional knockout leukemia-derived cell line infected with an empty vector (control) or with constructs overexpressing wild-type KAT7, K277Q mutant KAT7 or K277R mutant KAT7. C, Quantification of total cell numbers 6 days after 4-hydroxytamoxifen-induced loss of SIRT1 in cells overexpressing an empty vector or different KAT7 versions from B. D, Quantification of apoptosis (Annexin V-positive cells) 6 days after 4-hydroxytamoxifen-induced loss of SIRT1 in cells overexpressing an empty vector or different KAT7 versions from B. * P < 0.05 and ** P < 0.01 in Figs. 7C-D using 2-way analysis of variance (ANOVA) for multiple comparisons.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Knock-Out, Derivative Assay, In Vitro, Two Tailed Test, Western Blot, Infection, Plasmid Preparation, Construct, Mutagenesis

Journal: Haematologica

Article Title: BCAT1 is a NOTCH1 target and sustains the oncogenic function of NOTCH1

doi: 10.3324/haematol.2024.285552

Figure Lengend Snippet: BCAT1 is upregulated during NOTCH1-dependent transformation. (A) Heat map showing the top 50 most downregulated and upregulated genes between normal double-positive (DP) cells and ICN1 -induced DP leukemic cells (NIC Tumors). (B) Expression levels (quantitative polymerase chain reaction [qRT-PCR]; left) of Bcat1 in thymocytes obtained from 6-8-week-old C57/ Bl6 mice and leukemic cells from 6 AE-NOTCH1 T-cell acute lymphoblastic leu kemia (T-ALL) tumors (NOTCH1-T). Significance was calculated using an unpaired two-tailed t test. ** P <0.01. Western blot (right) showing protein expression levels of ICN1 and Bcat1. (3-actin and tubulin are shown as loading controls. Graphical representation of Bcat1/|3-actin ratios (extreme right). Bars represent mean values. ICN1: intracellular NOTCH1. (C) Box plot showing the expression of BCAT1 mRNA in T-ALL patients (N=57) and thymocyte subsets (7 thymocyte and mature T-cell subsets derived from [N=3] independent donors; quantile-normalized microarray results downloaded from GSE33469 and GSE33470). CD3 + and CD3- DP cells were grouped together. CD1 + and CD1-CD34 + cells were grouped together. Boxes represent first and third quartiles and line represents the median. Statistical analysis between groups was performed using unpaired two-sided t test. (D) BCAT1 transcript (top) and protein levels (bottom) in total human thymus, NOTCH1 wild-type and NOTCH1 -activated/mutated patient derived T-ALL patient-derived xenografts (PDX). Significance was calculated using a non-parametric t test (Mann-Whitney). ** P <0.01. ICN1, MYC and PTEN protein levels are also shown. (3-actin is shown as loading control. (E) PDX samples were treated in vivo with DBZ (10 μg/kg every 8 hours [h] for a total of 3 injections) or vehicle (dimethyl suldoxide [DMSO]) for 24 h before analysis of BCAT1 transcript levels. For statistical analysis, an unpaired t test was used. ** P <0.01, *** P <0.001. (F) NOTCH1 chromatin immunoprecipitation (ChIP)-sequencing binding (left) in the BCAT1 locus in HPB T-ALL cells. Inset shows the location of ChIP-quantitative polymerase chain reaction (qPCR) amplicons near NOTCH-1 peak region (P1-P2) and in a negative control region (NL). Chromatin from PF382 cells was subjected to ChIP using a NOTCH1 antibody (right). The indicated regions (P1, P2 and NL) were PCR amplified from the precipitated and input DNA. Fold enrichment was calculated as a ratio of amplification efficiency of ChIP sample over that of the immunoglobulin G (IgG) control. Shown are means ± standard deviation SD (N≥3). For statistical analysis, an unpaired t test was used. *** P <0.001. NS: not significant.

Article Snippet: Antibodies against tubulin (TU-02), MYC and p53 (DO-1) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing cleaved NOTCH-1 (ICN1; Val 1744), β-actin, p21, BCAT2, BCAT1, Ku80, Ku70, histone H3, cleaved PARP-1, cleaved caspase 3, phosphorylated H2AX (pS139), phosphorylated DNA-PKcs (pS2056), total DNA-PKcs, phosphorylated ATM (pS1981), total ATM, phosphorylated CHK2 (pT68), total CHK2, phosphorylated TP53 (pS15), acetylated p53 (K382) and GADPH were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Western Blot, Derivative Assay, Microarray, MANN-WHITNEY, Control, In Vivo, Chromatin Immunoprecipitation, ChIP-sequencing, Binding Assay, Negative Control, Amplification, Standard Deviation