Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: Northern blot analysis of murine T-ALL cell lines reveals 2 types of aberrant Notch1 transcripts. Northern blot analyses used polyA RNAs from normal murine thymus or the indicated cells lines. Panels A, B, and C correspond to Northern blots hybridized to probes A (exon 34), B (exon 26), or C (exon 1), which are homologous to the regions shown in the cartoon of the Notch1 locus above the blots. Numbers correspond to the position of RNA size markers in kilobases. FL indicates full-length Notch1 transcript. *Short Notch1 transcripts.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Northern Blot

Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: Structure of type 1 and type 2 Notch1 transcripts. (A) Products amplified from 144 cells, a line expressing type 1 Notch1 transcripts, using a 3′ exon 27 primer and 5′ linker primer, are shown after agarose gel electrophoresis. Sequences of 5′RACE products are shown below; the basepair numbering is in relationship to the A residue in the ATG start site in exon 1 of the Notch1 gene, which is designated position 1. (B) Northern blot analysis of lines expressing type 1 Notch1 transcripts with probes from the 3′ end of exon 25 and the 5′ end of exon 25. The diagram shows the regions of the Notch1 that are encoded by exons 25 and 27, respectively. (C) Sequences of RT-PCR products obtained from murine T-ALL cell lines expressing type II transcripts. Sequencing of products obtained from 130.1 and 130.2 cells revealed a transcript in which exon 1 is spliced out of frame to exon 27. In SCID-adh cells, a product was obtained in which exon 1 is spliced to an intron 1 sequence that contains an in-frame stop codon, which is in turned joined to an internal sequence within exon 28.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Amplification, Expressing, Agarose Gel Electrophoresis, Residue, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: Southern blot analysis reveals 2 types of 5′ Notch1 genomic rearrangements in murine cell lines expressing type 1 (I) or type 2 (II) aberrant transcripts. Genomic DNA (10 μg) from the indicated cell lines was digested with Eco R1 and analyzed on Southern blots. Panels A, B, and C correspond to Southern blots hybridized to probes A, B, and C, which are homologous to the regions shown in the diagram of the Notch1 locus above the blots. The positions of genomic Eco R1 sites (R1) around the Notch1 locus are expressed relative to the position of the ATG start codon in exon 1, which is designated position 1. The positions of murine-specific alternative 5′ Notch1 exons 1a, 1b, and 1c are also shown. R indicates rearranged; and GL, normal genomic locus.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Southern Blot, Expressing

Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: Evidence of RAG involvement in Notch1 rearrangements in murine T-ALL. (A) Rearrangements in Notch1 deduced from sequencing of PCR products generated from 11 cell lines with type 1 deletions (top) and 3 cell lines with type 2 deletions (bottom). GL is the sequence of the germline DNA flanking the breakpoints. Nucleotide positions are expressed relative to the ATG start codon in exon 1 of Notch1. Flanking sequences resembling RAG recognition sequences are boxed. Residues matching the consensus RAG signal sequence (a CACATGT heptamer followed by a 12- or 23-bp spacer and the nonameric sequence ACAAAAAAC) are denoted with an asterisk. N nucleotides and P nucleotides (underlined) are also shown. The point of joining in SCID-adh contains a single cytosine residue (underlined) of unknown origin. (B) Distribution of RAG2 binding and H3K4 trimethylation across the murine Notch1 locus. ChIP-Seq was performed with antibodies specific for RAG2 and H3K4-me3 on DNA immunoprecipitated from normal thymocytes (αRAG2 WT), thymocytes expressing a RAG1 D708A mutant (αRAG2 Mut) that binds chromatin but is catalytically inactive, and homozygous RAG2 knockout thymoctyes (αRAG2−/−).22 Histograms showing sequence reads that aligned to the murine genome are superimposed on a diagram of the Notch1 locus. The y-axis of each histogram corresponds to the number of aligned reads per 106 total reads.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Sequencing, Generated, Residue, Binding Assay, ChIP-sequencing, Immunoprecipitation, Expressing, Mutagenesis, Knock-Out

Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: M1727 serves as the translational start site in aberrant type 1 and type 2 Notch1 transcripts. (A) Schematic showing the positions of codons M1616 (M1), M1659 (M2), and M1727 (M3) within type 1 and type 2 Notch1 transcripts relative to the full-length Notch1 polypeptide. (B) Western blot analysis of 293 cells transiently transfected with empty pcDNA3, type 1 transcript cDNA (corresponding to nucleotides 4438-7665 of murine Notch1), type 1 transcript cDNAs bearing M to L point mutations in codons 1616, 1659, or 1727, type 2 transcript cDNA from SCID.adh cells, or type 2 transcript cDNA with an M to L point mutation in codon 1727. Blots were stained with antibodies specific for activated Notch1 (V1744 antibody, Cell Signaling Technology) or β-actin. (C) Notch reporter gene studies conducted in U2OS cells transiently transfected in triplicate with equivalent amounts of a CSLx4 firefly luciferase reporter gene, an internal Renilla luciferase control gene, and empty pcDNA3 vector or pcDNA3 vectors containing the indicated cDNAs. The left-hand panel compares the activities of a murine type 1 Notch1 transcript; a murine type 1 Notch1 transcript containing a M1727L mutation; a murine type 1 Notch1 transcript containing a stop mutation introduced at codon 2415 (ΔP) that deletes the C-terminal 116 amino acids of murine Notch1; a full-length human NOTCH1 cDNA containing a weakly activating NRR mutation, L1601P, with and without a ΔP frameshift mutation at codon 2473 that deletes the C-terminal 83 amino acids of human Notch1; and a full-length human NOTCH1 cDNA containing a strongly activating in-frame insertion in the NRR designated P12, with and without a ΔP frameshift mutation at codon 2473. The right-hand panel compares the activities of murine type 1 and type 2 transcripts with a human NOTCH1 cDNA bearing a L1601P deletion. Each cDNA was tested in the presence and absence vehicle (dimethyl sulfoxide [DMSO]) or vehicle plus the GSI DAPT (10μM). After normalization to an internal Renilla luciferase control, firefly luciferase activities were expressed relative to the empty vector control, which was arbitrarily set to 1. Error bars represent SD. The results shown are representative of 2 independent experiments.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Western Blot, Transfection, Mutagenesis, Staining, Luciferase, Control, Plasmid Preparation

Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: Detection of 5′ deletions and aberrant Notch1 transcripts in primary murine “thymomas.” (A) Sequences of PCR products obtained by amplification of genomic DNA isolated from 2 thymic lymphomas with primers flanking the most common breakpoints associated with type 1 aberrant transcripts. Sites of DNA breakage and joining, as deduced from sequencing of PCR products, are shown. Residues matching the consensus RAG recognition sequence (CACAGTG followed by a 12- or 23-bp spacer and the sequence ACAAAAAAC) are denoted with an asterisk. N nucleotides and P nucleotides (underlined) are also shown. GL indicates germline DNA flanking the breakpoints. Boxes represent sequences resembling RAG signal sequences. (B) Ratiometric Notch1 quantitative RT-PCR analysis. The relative amounts of transcripts containing 5′ (exons 23 and 24) and 3′ (exons 30 and 31) Notch1 sequences were determined for the tumors in panel A and normal murine thymus, a cell line with a homozygous type 2 deletion (135.2), and a cell line with a heterozygous type 1 deletion (144). Each determination was made in triplicate. The results shown are representative of 2 independent experiments.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Amplification, Isolation, Sequencing, Quantitative RT-PCR

Journal: Blood

Article Title: Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

doi: 10.1182/blood-2010-05-286328

Figure Lengend Snippet: Mechanisms of ligand-independent ICN1 production in T-ALLs bearing Notch1 deletions. (A-B) Structure and functional consequences of type 1 and type 2 Notch1 deletions. (C) Conservation of M1727 in vertebrate Notch1 receptors. *Identical residues. “:” indicates conserved residues. TM indicates transmembrane domain; S3, site of intramembranous γ-secretase cleavage; and ICN, intracellular Notch.

Article Snippet: Cell growth assays Approximately 1 × 10 4 cells/well in 96-well plates were cultured in the presence of human IgG (10 μg/mL), anti-Notch1 inhibitory antibodies (10 μg/mL), or 1μM compound E (Tocris).

Techniques: Functional 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: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Restoration of NOTCH1 signaling inhibits in vitro growth and alters morphology in NOTCH1 mutant HNSCC tumors. A . Ectopic expression of WT NFL1 leads to activation and cleavage of NOTCH1 (Cl-NOTCH1) protein in 3 different NOTCH1-mutant HNSCC cell lines that increases upon 16 h stimulation with immobilized JAG1 ligand, compared to control FC protein. B . Continued growth on immobilized JAG1 for 8-12 days inhibits colony formation in NOTCH1-mutant cell lines with restored NFL1. C . Quantitation of NFL1-mediated growth inhibition of colonies. D. Restoration of NFL1 signaling in UMSCC-22A led to dramatic reduction in cell size and formation of loosely attached tumor spheroids after 5 days only in the presence of JAG1. UM47 = UMSCC47, UM22A = UMSCC22A

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: In Vitro, Mutagenesis, Expressing, Activation Assay, Control, Quantitation Assay, Inhibition

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Restoration of NOTCH1 signaling induces senescence. A. Photomicrographs of NOTCH1 -mutant HNSCC cell lines infected with NFL1 or empty MigR1 grown on either JAG1 or FC control protein and stained for β-gal after 5 to 7 days culture. B. Quantitation shows significant elevation of β-gal staining in the presence of NFL1 expression and growth on JAG1 ligand.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Mutagenesis, Infection, Control, Staining, Quantitation Assay, Expressing

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: NOTCH signaling alters cell morphology and inhibits growth in two-dimensional cultures. A. Expression of full length transmembrane NOTCH1 (Tm-NOTCH1) in cells with WT NOTCH1 (PJ34, 183, CAL27, and UMSCC1) and cleaved activated NOTCH1 (cl-NOTCH1) protein after 16 h growth on immobilized Notch1 ligand (JAG1) or FC control protein (FC). B. Extended growth (e.g. 8-12 days) on JAG1 ligand significantly reduced colony formation compared to control FC protein in four NOTCH1 WT cell lines. C. Growth on JAG1, but not control FC induces morphological transformation of cell lines with WT NOTCH1 (183 and PJ34) observed by 5 days, characterized by reduced cell size and formation of loosely attached tumor spheroids. D. Tumor spheroids induced by growth on JAG1 express the senescence marker b-gal. E. Ectopic expression of dominant negative MAML1, which inhibits NOTCH1 mediated transcriptional regulation, prevents JAG1-induced tumor spheroid formation and reverses inhibition of colon formation in PJ34 cells.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Expressing, Control, Transformation Assay, Marker, Dominant Negative Mutation, Inhibition

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Both NOTCH1 and NOTCH2 signaling contribute to JAG1-induced growth inhibition but NOTCH1 signaling is sufficient. A. CRISPR KO of either NOTCH1 (NIKO), NOTCH2 (N2KO) or double KO (N1N2 KO) in PJ34 were validated by western blotting for total NOTCH1 and NOTCH2. Appearance of cl-NOTCH1was absent in N1KO or N1N2KO after stimulation with JAG1 for 16 h. B. N1KO or N2KO partially blocked JAG1-induced inhibition of colony formation, while double N1K2 KO completely prevented growth inhibition. C. Infection with ectopic NFL1 cDNA restored NOTCH1 signaling in N1N2 KO cells. D. Re-Expression of NFL1 in PJ34 after N1N2KO restored JAG1-mediated inhibition of colony growth. E. Loss of both NOTCH1 and NOTCH2 (N1N2KO ) in PJ34 protected cells from JAG1-induced morphology changes while re-expression of NFL1 restored spheroid formation caused by growth on JAG1.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Inhibition, CRISPR, Western Blot, Infection, Expressing

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: RPPA identifies NOTCH1 WT cell lines with elevated baseline cl-NOTCH1expression which was inversely correlated with protein expression of AXL and a-CATULIN. A. Normalized RPPA values of cl-NOTCH1measured from NOTCH1 mutant and NOTCH1 WT HNSCC cell lines. Cell lines where cl-NOTCH1 protein was validated by western blots (WB) are indicated by colored symbols, with red dots for NOTCH1 mutants (HN31, UMSCC22A, UMSCC47, and HN4), light blue dots for NOTCH1 WT cells with low to moderate baseline cl-NOTCH (PJ34, 183, CAL27, and UMSCC1), and dark blue dots corresponding to NOTCH1 WT cells with high basal cl-NOTCH1 (FaDu, PCI24, SCC61, SCC15, and MDA1986LN). B. Western blot confirmation of high cl-NOTCH1protein in subsets of WT cell lines, compared to PJ34 with low baseline activation. C. Confirmation of high baseline NOTCH1 activation in MDA686LN. D . DBZ inhibited cl-NOTCH1 protein which was associated with increased levels of both AXL and a-CATULIN protein.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Expressing, Mutagenesis, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: NOTCH1 is not a driver of cell growth in multiple HNSCC cell lines with high endogenous NOTCH1 activation. A. Western blot validation of high basal levels of cl-NOTCH1protein in 5 NOTCH1 WT HNSCC cell lines with different genomic backgrounds and persistent inhibition of NOTCH1 signaling after 72 h treatment with various doses of the NOTCH1 inhibitor DBZ. B . Staining of colony formation in the presence or absence of continuous treatment with DBZ inhibitor (replaced every 48 h) for the duration of culture. C. Quantitation of colony formation shows no difference in growth after continued treatment with DBZ, except for FaDu cells which grew slightly better after inhibition of NOTCH1 signaling.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Western Blot, Biomarker Discovery, Inhibition, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: NOTCH1 regulates genes involved in proliferation, differentiation, attachment, motility, and response to oxygen containing compounds. The top 120 significant genes (i.e., minimum of 1.4-fold change) from NOTCH1 WT PJ34 and 183 cells regulated in common by growth on JAG1 and their biological pathways. The fold change in each cell line after growth for 5 days on JAG1 is annotated vertically as a color gradient with red boxes representing increases, and blue boxes representing decreases. Membership in select GO pathways that were enriched in the gene set is annotated with black boxes, and gold boxes indicate genes that were kept for the final NOTCH1 gene activation signature discussed in the text.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: The activation of NOTCH1 signaling through multiple experimental approaches consistently downregulates oncogenic drivers and molecules mediating cell adhesion. A. Growth for 5 days on JAG1 significantly inhibited RNA expression of the basal cell marker genes ITGA1, ITGA3, ITGA6, LAMC1, and LAMC2 in two NOTCH1 WT cell lines PJ34 and 183, while stimulating expression of the suprabasal maker genes KRT4and KRT13. B. Ectopic expression of cDNA encoding cl-NOTCH1decreased expression of ITGA3, LAMC2, AXL, and α-CATULIN at 3 days and 5-days post infection in a NOTCH1 WT cell (PJ34) and in a NOTCH1 -mutant cell (HN31). C . AXL and a-CATULIN protein levels decline in NOTCH1 WT (PJ34) tumor cells grown on JAG1 (3 days) and in 3 different NOTCH1 mutant cell lines (HN31, UMSCC22A, UMSCC47) when expression of WT full length NOTCH1 receptor (NFL1) is restored (NFL1) and then grown on JAG1 ligand for 3 days. Cells were also infected with empty vector (MigR1) and/or grown on control FC protein. D. PJ34 with CRISPR-mediated NOTCH1 KO were further engineered to express a DOX inducible cl-NOTCH cDNA (PJ34-iICN1) whose expression was titrated after 72 h treatment with different doses of DOX, to achieve protein levels equivalent to parental PJ34 stimulated with JAG1 for 16 hr. E. ICN1 induction with 1000 ng/ml DOX caused cells to massively shrink in size and form loosely attached tumor spheroids. F. Venn diagram illustrates overlap of genes in PJ34 significantly upregulated (FDR <0.1, |Fold change| ≥ 1.25-fold) by growth on JAG1 or after 24 h iICN1 induction, and genes specifically bound in the promoter or gene body regions by iICN1 after Chip-seq experiments. G. Venn diagram illustrating overlap of genes in PJ34 significantly downregulated (FDR <0.1, |Fold change| ≥ 1.25-fold) by growth on JAG1 or after 24 h iICN1 induction, and genes specifically bound in the promoter or gene body regions by iICN1 after Chip-seq experiments.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, RNA Expression, Marker, Expressing, Infection, Mutagenesis, Plasmid Preparation, Control, CRISPR, ChIP-sequencing

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Ectopic expression of ICN1 mimics changes induced by JAG1 stimulation. A. Infection with ICN1 but not empty vector (MigR1) strongly inhibited protein expression of AXL and α-CATULIN. Widely used ICN1 cDNA product which begins several amino acids downstream of the native cleavage site is recognized by antibodies to the C-terminal region of NOTCH1 but not antibodies specific to the cleavage site. B. Expression of ICN1 in NOTCH1 mutant UMSCC22A produced the same morphological changes observed earlier upon expression of NFL1 and growth on JAG1. C. Expression of ICN1 in NOTCH1 WT 183 produced the same morphological changes observed earlier with growth on JAG1. D. Expression of ICN1 but not MigR1 control in NOTCH1 WT PJ34 produced the same morphological changes observed earlier with growth on JAG1. E. ICN1 expression triggered decreased protein expression of AXL, α-CATULIN, and ITGA3 in 183 cells.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Expressing, Infection, Plasmid Preparation, Mutagenesis, Produced, Control

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Evidence of NOTCH1 signaling in a subset of HNSCC tumor cell lines. Protein levels of both AXL and α-CATULIN increase when activated cl-NOTCH1 formation is blocked with the NOTCH inhibitor DBZ in five different NOTCH1 WT cell lines with high endogenous NOTCH1 activity.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Physiological levels of iICN1 trigger morphology changes in NOTCH1 WT and mutant tumor cell lines. A. Titration demonstrates that treating FaDu-iICN1 with 250ng-500 ng/ml DOX for 36 h induces protein levels of iICN1 equivalent to stimulating PJ34 with JAG1 for 16 h. B . As little as 300 ng/ml DOX for 6 days causes tumor spheroid formation in FaDu-iICN1. C. cl-NOTCH 1protein levels in UMSCC22A-iICN1 treated with 200-300 ng/ml DOX for 36 h are similar to basal levels in FaDu. D. Tumor spheroid formation in UMSCC22A-iICN1 cells after 6 days treatment with 100 ng/ml DOX.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Mutagenesis, Titration

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Dose response inhibition of colony formation by iICN1 in NOTCH1 mutant and WT cell lines. A. Crystal violet stain of colonies after increasing doses of DOX. B . Quantitation of colony inhibition after treating cells with increasing DOX doses. C. Treatment of control Tet3G expressing cell lines, lacking an iICN1 construct, with 1000 μg/ml DOX did not inhibit colony growth.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Inhibition, Mutagenesis, Staining, Quantitation Assay, Control, Expressing, Construct

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: NOTCH1 activation increases tumor spheroid growth and anoikis resistance. A. Addition of 1000 mg/ml DOX increases the number of orospheres derived from FaDu-iICN1 in non-adherent low serum concentrations for 1 week. B . Increased orosphere numbers after growing UMSCC22A-iICN1 in 300 mg/ml DOX non-adherent low serum cultures for 1 week. C. DOX was added to attached UMSCC22A-iCN1 at 300 mg/ml, FaDU-iICN1 at 1000 mg/ml, or control Tet3G cells for 48 h before single cell suspensions were prepared in low serum containing media and an rotated in non-adherent tubes inside a humidified 37 0 C cell incubator for an additional 30 hours, in continued presence or absence of DOX. Viability of replicate cultures was examined with an automated cell counter.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Activation of NOTCH1 does not increase the percentage of CD44+bright ALDEFLUOR-positive cells in NOTCH1 mutant or WT tumor lines. A. Flow cytometry gating strategy to identify live single cells. B. Representative gating of UMSCC22A-iICN1 cells in absence of Dox. The sample is split and one tube treated with a BV-421 conjugated isotype control antibody and the aldehyde dehydrogenase inhibitor (DEAB) before adding fluorescein conjugated substrate to set the background gates (left panel). The second tube is incubated with BV421-A conjugated anti-CD44 and Aldefluor substrate without DEAB to gate on the CD44+/Aldefulor+ population (middle panel) and a single fixed threshold is set for the top 10% brightest CD44+ (e.g., CD44+ bright), which is uniformly applied to all remaining UMSCC22A-iICN1 samples. The CD44+ bright population is further analyzed for the percentage of Aldefluor+ cells (right panel). C. A UMSCC22A-iICN1 sample pretreated with 200 ng/ml DOX for 48 h to induce ICN1 before staining is gated in the same fashion, except Aldefluor gating is set with a sample specific tube treated with DEAB (left panel) and CD44+bright cells are analyzed in a companion tube without DEAB for percentage of Aldefulor+ using the previously set CD44+ threshold (right panel). D. The total percent of CD44+bright/Aldefluor+ cells does not increase after iICN1 induction in either UMSCC22A-iICN1 or FaDu-iICN1.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Mutagenesis, Flow Cytometry, Control, Incubation, Staining

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Activation of NOTCH1 fails to increase surface CD133+ but increases expression of SOX2 in NOTCH1 mutant (UMSCC22A-iICN1), or NOTCH1 WT cell lines (FaDu-iICN1 and PJ34-iICN1). A. Cells were incubated for 48 h with or without DOX at 200 ng/ml (UMSCC22A), 300 ng/ml (FaDu), or 1000 ng/ml (PJ34) before staining with antibody to surface CD133 by flow cytometry. Fluorescent intensity histograms are shown for control biological replicates without DOX (red and light blue traces) or after DOX treatment (green and orange traces). B. Statistical comparison of CD133 mean fluorescence intensity (MFI). C. Western blot analysis of SOX2 protein expression in similarly treated cells.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Expressing, Mutagenesis, Incubation, Staining, Flow Cytometry, Control, Comparison, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: NOTCH1 activation reduces in vivo tumor growth and formation in NOTCH1 WT FaDU. After CRISPR-mediated NOTCH1 KO, FaDu were engineered to express iICN1 and pretreated with or without 300 ng/ml DOX for 72 hours in vitro before injecting 100 cells (A), 1000 cells (B), 10,000 cells (C), or 100,000 cells (D) into flanks of mice. The DOX treated group received additional in vivo DOX (1 mg) for one week by oral gavage after implantation and tumor growth was plotted verses post-inoculation time. Time to tumor formation in mice was plotted verses post-inoculation time for 100, 1000, 10,000, or 100,000 inoculated tumor cells (E-F) in the no Dox (grey) or DOX (black) treated groups.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, In Vivo, CRISPR, In Vitro

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Activation of NOTCH1 signaling in NOTCH1 mutant tumors profoundly inhibits vivo tumor growth and reduces the number of CAF cells. A. UMSCC22A-iICN1 was inoculated into mouse flanks and mice were randomly assigned to receive either no DOX or daily DOX (1 mg) by oral gavage for two weeks. Tumor volumes were compared over time. B . H&E staining of a representative tumor that eventually formed in mice treated with DOX (top panel) and a tumor that formed in the absence of DO X (bottom panel). Filled arrows indicate tumor nests, while hallow arrows designated areas predominately populated by CAFs. The percentage of CAFS in tumors was significantly lower when ICN1 was induced with DOX.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Mutagenesis, Staining

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Knockdown of AXL and a-CATULIN to mirror NOTCH1 signaling inhibits in vitro and in vivo tumor growth. A. Western blot confirmation of AXL and a-CATULIN protein inhibition following shRNA KD in NOTCH1 mutant HN31 cells 96 h post-infection with specific shRNA. B. Western blot confirmation of AXL and a-CATULIN protein inhibition following shRNA KD in NOTCH1 WT PJ34 cells 96 h post-infection with specific shRNA. C. KD of AXL and a-CATULIN blocks in vitro colony formation in HN31. D. KD of AXL and a-CATULIN blocks in vitro colony formation in PJ34. E. KD of AXL and a-CATULLIN profoundly inhibits HN31 in vivo tumor growth in an orthotopic tongue tumor model. E. E. KD of AXL and a-CATULLIN with alternate shRNA vectors targeting different regions inhibits HN31 in vivo tumor growth in a subcutaneous flank model.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Inhibition, shRNA, Mutagenesis, Infection

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Development of an in vivo NOTCH1 signaling gene expression signature. A. The TCGA OCSCC RNA-seq cohort was mined for cross-correlation of RNA expression using gene candidates from the list of 119 top genes regulated in vitro after JAG1 exposure. Hierarchical two-way clustering of cross-correlation coefficients identified two main gene clusters annotated by whether the genes were up-regulated (red box) or down regulated (blue box) after JAG1 exposure. B. Re-clustering TCGA OCSCC cross-correlation coefficients with the remaining 95 genes after removal of those with inconsistent in vitro and in vivo behavior.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: In Vivo, Gene Expression, RNA Sequencing, RNA Expression, In Vitro

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Selection of optimal sample clusters. Consensus hierarchical clustering of TCGA OCSCC and LHSCC samples using the 95 gene NOTCH1 signature was performed and the similarity matrices achieved using increasing numbers of sample clusters (e.g. N=1 to 4) were compared to theoretical perfection matrices to select a local minimum for the normalized Euclidean distances (N.E.D.) to identify an optimal number of sample clusters, which happened to be N =2 clusters for each disease subsite.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Selection

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Comparison of genes regulated by NOTCH1 in vitro and genes differentially expressed in primary HNSCC tumors with a NOTCH1 activation signature. A . Consensus hierarchical clustering of TCGA OCSCC specimens based on a 95-gene NOTCH1 activation signature identified a cluster of patients (Cluster 2, N = 57) with an expression pattern indicative of active NOTCH1 signaling and another cluster (Cluster 1, N = 255) predicted to have loss of NOTCH1 signaling. Genes are annotated with vertical boxes according to whether they were upregulated (red) or downregulated (blue) by JAG1 in vitro. Samples with a NOTCH1 mutation are annotated horizontally with a black box and association between NOTCH1 mutation status and cluster for patients with sequencing information was analyzed by Chi-square analysis. B. Parallel clustering and analysis of TCGA LHSCC samples using the same 95-gene NOTCH1 signature. C. Venn diagram illustrating overlap of genes upregulated (FDR<0.1, |FC|≥1.25) by NOTCH1 in vitro (JAG_up), the subset of upregulated genes part of the NOTCH1 signature (Sig_up), and genes upregulated (FDR<0.1, |FC|≥1.25) in Cluster 2 from OCSCC or LHSCC. D. Venn diagram illustrating overlap of genes downregulated (FDR<0.1, |FC|≥1.25) by NOTCH1 in vitro (JAG_down), the subset of downregulated genes part of the NOTCH1 signature (Sig_down), and genes downregulated (FDR<0.1, |FC|≥1.25) in Cluster 2 from OCSCC or LHSCC.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Comparison, In Vitro, Activation Assay, Expressing, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: NOTCH1 activation in primary HNSCC is associated with changes to the tumor microenvironment. A. ssGSEA scores representing 18 different leukocyte subsets derived from TCGA OCSCC were used for hierarchical clustering to classify samples as immunologically cold (sample cluster 1) or hot (sample cluster 3) and the membership of samples from previous clustering based on the NOTCH1 gene signature is annotated with a black box for NOTCH1 signaling on or a grey box for NOTCH1 signaling off. B. NRF2 pathway scores are compared across NOTCH1 sample clusters from OCSCC and LHSCC TCGA tumors. C. CAF pathway scores, indictive of CAF infiltration, are compared across NOTCH1 sample clusters from OCSCC and LHSCC TCGA tumors.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Derivative Assay

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Visual confirmation that TCGA tumor samples identified with active NOTCH1 signaling contain fewer CAF. H&E images were downloaded from the TCGA data portal and the CAF ssGSEA scores appear in parenthesis alongside the sample TCGA identification numbers.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques:

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Higher levels of NOTCH1 activation correlate with better survival in OCSCC and LHSCC TCGA cohorts. A. No difference in OS among TCGA OCSCC patients when NOTCH1 activation is treated as a categorical variable based on clusters with the NOTCH gene signature. B . Validation that ssGSEA scores derived from the NOTCH1 gene signature provide a continuous value measurement that faithfully replicates sample clustering. C. OCSCC samples with higher ssGSEA scores above a threshold (−1554) determined by recursive partitioning have significantly improved OS. D. LHSCC samples with higher ssGSEA scores above the same threshold (−1554) have significantly improved OS, validating the threshold. E. OCSCC patients with higher NOTCH1 signaling have improved PFS. F. LHSCC patients with higher NOTCH1 signaling have improved PFS.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques: Activation Assay, Biomarker Discovery, Derivative Assay

Journal: bioRxiv

Article Title: NOTCH1 Acts as a Tumor Suppressor That Induces Early Differentiation in Head and Neck Cancer

doi: 10.1101/2025.04.25.650710

Figure Lengend Snippet: Patients lacking NOTCH1 mutations are significantly enriched for genomic alterations in PIK3CA. The proportion of tumors with genomic alterations in PIK3CA, including mutations and/or high-level gene copy gains, is significantly higher in in the group associated with NOTCH1 signaling identified through hierarchical clustering of TCGA OCSCC (A) or (B) LHSCC.

Article Snippet: Antibodies to activated cleaved NOTCH1 (cl-NOTCH1), total NOTCH1, total NOTCH2, AXL, FLAG tag, were from Cell Signaling; whereas antibodies to α-CATULIN LAMC2, ITGA3, ITGA5 were from Santa Cruz and anti-Hes5 was from Abcam.

Techniques:

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: 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: 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: Subsequently membranes were incubated with corresponding primary antibodies in 5% milk TBST overnight at 4 °C (Rabbit recombinant anti-GUCY1B1 antibody 1:1000 (Abcam, 154841), Rabbit phospho-VASP (Ser239) antibody 1:1000 (Cell Signaling Technology, 3114, RRID:AB_2213396), Rabbit anti-VASP Antibody 1:5000 (Bethyl Laboratories, A304-769A-M, RRID:AB_2782159), Rabbit anti-Synaptophysin antibody 1:20,000 (Abcam, ab32127, RRID:AB_2286949), Rabbit HES1 (D6P2U) mAb 1:500 (Cell Signaling Technology, 11988, RRID:AB_2728766), Rabbit GAPDH (14C10) mAb 1:5000 (Cell Signaling Technology, 2118, RRID:AB_561053), Rabbit Notch1 Antibody 1:500 (Bethyl Laboratories, A301-895A, RRID:AB_1524102), Rabbit Histone H3 Antibody 1:5000 (Cell Signaling Technology, 9715, RRID:AB_331563), Rabbit α-Tubulin Antibody 1:5000 (Cell Signaling Technology, 2144, RRID:AB_2210548), Rabbit Vinculin Antibody 1:20,000 (Abcam, ab129002, RRID:AB_11144129)) and the appropriate horseradish peroxidase-coupled secondary IgG 1:10,000 (Agilent, P044801-2, RRID:AB_2617138) in 5% milk TBST for 1 h at room temperature.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Ex Vivo, ChIP-qPCR, Binding Assay, Positive Control, Negative 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 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: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Activated NOTCH1 is detected in valve cells in a flow dependent manner. (A) Normal human aortic valve section stained with an antibody specific to the active form of NOTCH1 (NICD, red). NICD was found in both endothelial (arrows) and interstitial cells (arrowheads). Autofluorescence (AutoFI, green) of collagen and elastin highlight the fibrosa layer (F) and ventricularis layer (V), respectively. Nuclei, DAPI (blue). Scale bars indicate 100 μm. (B) Schematic of experimental procedure. Normal HAVECs were transfected with control or NOTCH1 siRNA, then cultured in static or fluid flow conditions. Gene expression was compared by qRT-PCR or mRNA-seq. (C) qRT-PCR analysis of HAVECs from four conditions. NOTCH1, two canonical direct targets, HES1 and HEY2 and a known flow responsive gene, KLF2, were analyzed. Graphs show mean gene expression relative to the static, control siRNA condition with error bars representing standard deviation. (n=3; *, p<0.05; ** p<0.01; *** p<0.001; NS, Not Significant).

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Staining, Transfection, Cell Culture, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Pathways Activated by Shear Stress in NOTCH1 Dependent Manner

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Activation Assay, Coagulation

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Expression of endochondral ossification genes is affected by shear stress and NOTCH1 signaling. Graphs show mean gene expression relative to the static, control siRNA condition with error bars representing standard deviation. All numbers are shown in log2 scale. (n=3; *, p< 0.05; **, p< 0.01; ***, p< 0.001, NS, Not Significant).

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Expressing, Standard Deviation

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Heatmap and clustering analysis of RNA-seq and ChIP-seq from HAVECs reveal likely NOTCH1 direct targets. (A) The expression of each gene was normalized to the static, control siRNA condition and then Log2 transformed. Displayed genes were selected as matching one of two patterns reflecting coordinate regulation by both NOTCH1 and shear stress: Pattern 1) at least 2-fold down upon NOTCH1 siRNA knockdown in both static and flow conditions, and at least 2-fold up in flow control siRNA condition compared to static, or Pattern 2) at least 2 fold up in NOTCH1 siRNA knockdown in both static and flow conditions, and at least 2 fold down in flow control siRNA condition compared to static. (B) ChIP-seq score shows the value for the highest ChIP peak +/− 20 kb from the transcriptional start site (TSS) of each gene. Annotated genes represent potential direct targets of NOTCH1 based on ChIP-seq score.

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Expressing, Transformation Assay

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: MGP expression is NOTCH1 dependent. (A) Normal human aortic valve sections stained with antibodies specific to: i. cMGP, the active, carboxylated form of MGP; ii. ucMGP, the inactive, uncarboxylated form of MGP; and iii. phosphoSMAD 1/5/8 (pSMAD1/5/8) cMGP, ucMGP and pSMAD1/5/8 (red) were found in both endothelial (arrows) and interstitial (arrowheads) cells. Autofluorescence (AutoFI, green) of collagen and elastin highlight the fibrosa layer (F) and ventricularis layer (V), respectively. Nuclei, DAPI (blue). Scale bars indicate 100 μm. (B) qRT-PCR analysis of MGP mRNA expression in HAVECs under four conditions. Graph shows mean gene expression relative to the no flow, no siRNA condition with error bars representing the standard deviation. (n=3; *, p< 0.05). (C) Western blot detecting NICD, active MGP (cMGP) and GAPDH from HAECs infected in vitro with increasing amounts of myc-tagged NOTCH1 intracellular domain (NICD). (D) Immunostaining for NICD (red) in cultured Human Aortic Endothelial Cells (HAEC) or Human Aortic Interstitial Cells (HAIC). (E) qRT-PCR to measure MGP mRNA in control or NOTCHI-overexpressing HAICs. (F) Western blot detecting active MGP in control or γ-secretase inhibitor (DAPT) treated HAECs. (G) Quantification of cMGP protein in (F) normalized to Actin. Error bars show standard deviation (n=3; **, p< 0.01; ***, p< 0.001; NS, Not Significant).

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Expressing, Staining, Quantitative RT-PCR, Standard Deviation, Western Blot, Infection, In Vitro, Immunostaining, Cell Culture

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: NOTCH directly regulates MGP through an endothelial enhancer. (A) NICD1-myc ChIP-qPCR. Relative enrichment is shown for four sites: validated HES1 NOTCH1/CSL binding site, validated HES1 non-CSL binding site, predicted CSL binding sites within the 821 bp putative MGP enhancer and an MGP non-CSL site. The HES1 and MGP non-CSL binding sites are located approximately 2 kb from the CSL binding sites. Error bars indicate standard deviation. (n=4; *, p< 0.05). (B): EMSA snowing CSL binding to putative MGP enhancer. A 46 bp oligo corresponding to the NICD1-myc ChIP-seq peak was labeled with 32P-dCTP (MGP Probe). Incubation with in vitro transcribed and translated CSL (CSL TnT) shifted the probe (arrow) This interaction could be competed by addition of unlabeled MGP probe (Unlabeled MGP) or oligo corresponding to a validated HES1 enhancer containing CSL sites (Unlabeled HES1), but not with unlabeled oligos in which the CSL sites were mutated (Mut), demonstrating specificity. Reticulocyte lysate (Empty TnT) caused a higher, non-specific shift. (C) Images of whole mount or histological sections from E16.0 transgenic embryos containing the 821 bp MGP enhancer upstream of the Hsp68 minimal promoter driving LacZ with or without mutation of the three predicted CSL binding sites. Mice containing the wildtype (Wt) enhancer had strong endothelial cell expression of LacZ (blue) in the large vessels of the arterial system (8/13) and some weak expression in the aortic valve (arrows) (4/13 embryos). Mutation of CSL sites (Mut) abolished endothelial expression (8/8); the mutant enhancer had weak expression in the smooth muscle layer (arrowhead) of 3/8 embryos. Sections were counterstained with Eosin (red). Aorta, Ao; branchiocephalic artery, BC; left common carotid, LCC; aortic valve, AoV.

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: Binding Assay, Standard Deviation, ChIP-sequencing, Labeling, Incubation, In Vitro, Transgenic Assay, Mutagenesis, Expressing

Journal: Journal of molecular and cellular cardiology

Article Title: NOTCH1 Regulates Matrix Gla Protein and Calcification Gene Networks in Human Valve Endothelium

doi: 10.1016/j.yjmcc.2015.04.006

Figure Lengend Snippet: Model of NOTCH1 regulation of human endothelial cell calcification. Yellow shapes indicate different classes of molecules and their sub-cellular localization. Genes in black are regulated by shear stress and NOTCH1 signaling, while those in orange also have a significant NOTCH1 ChIP-seq peak within 20 kb of the transcriptional start site, suggesting potential direct transcriptional regulation by NOTCH1.

Article Snippet: The siRNAs used were NOTCH1 siRNA (cat# 4392422 s9633 and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies).

Techniques: ChIP-sequencing

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 is required for TAL1 binding to the Myb enhancers and for the retention of active chromatin marks. (A) H3K27ac, TAL1, and RUNX1 enrichment at the MYB locus by chromatin immunoprecipitation sequencing is shown in genome browser tracks (genome.ucsc.edu, human hg19). (B) The mouse genomic region (mm10) around the Myb locus is shown, depicting the E-BOX (TAL1)- and RUNX-binding sites at positions +15 kb and −92 kb from the Myb TSS. (C-D) Enrichment of RUNX1, TAL1, H3K27ac, and H3 to the +15-kb (C) and −92-kb (D) Myb enhancer regions determined by ChIP-qPCR in control or Runx1-deleted mouse T-ALL cells. Data are shown as the mean of 3 or 4 independent experiments with error bars representing ± 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-sequencing, ChIP-qPCR, Control

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 mediates survival in a TAL1-negative, TLX3-transformed human T-ALL cell line, and RUNX3 also contributes to human T-ALL survival. (A) The human T-ALL cell line HPB-ALL was transduced with lentiviruses expressing shRNAs against GFP or RUNX1. Apoptotic cells were quantified by Annexin V/7AAD staining followed by flow cytometry. Data are shown as the mean of 3 independent experiments with error bars representing ± SEM. (B) Gene expression in control or RUNX1 knockdown cells was determined by qRT-PCR. Three independent experiments were performed, and data are shown as means with error bars representing ± SEM. (C) RUNX1 or RUNX3 knockdown in KOPTK1 cells induces cell death. The human T-ALL KOPTK1 cell line was transduced with lentiviruses expressing shRNAs against GFP, RUNX1, or RUNX3. Apoptotic cells were quantified by Annexin V/7AAD staining followed by flow cytometry. (D) RUNX1 and RUNX3 binding to N-Me, MYB +14-kb, and MYB –93-kb enhancer loci was determined by ChIP-qPCR. Data are shown as the mean of 4 independent experiments with error bars representing ± SEM (*P < .05, multiple Student t tests). (E-F) The expression of MYB and MYC in RUNX1- or RUNX3-silenced KOPTK1 cells was determined by qRT-PCR. Data are shown as the mean of 3 or 4 independent experiments with error bars representing ± SEM. *P <.05; **P < .005; ***P < .0005, 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: Transformation Assay, Transduction, Expressing, Staining, Flow Cytometry, Gene Expression, Control, Knockdown, Quantitative RT-PCR, Binding Assay, ChIP-qPCR

Journal: Biology Open

Article Title: PRDM14 promotes RAG-dependent Notch1 driver mutations in mouse T-ALL

doi: 10.1242/bio.017699

Figure Lengend Snippet: Notch1 is mutated and bears focal epigenetic changes in PRDM14-expressing cells. (A) Upper, schematic of the Notch1 locus. Exons are shown as black boxes. The region deleted in PRDM14-induced tumors, the canonical transcription start site (5′ TSS), the downstream 3′ TSS, and the putative PRDM14 binding site are indicated. Lower, alignment of sequences from PRDM14-induced tumors with the consensus RSS and cryptic RSS (cRSS) found at Notch1 . Conserved nucleotides are shown in red. (B) Ratiometric qRT-PCR using primers spanning Notch1 exon 23-24 and Notch1 exon 30-31 in wild-type (WT) ( n =5) and R26PR;cre ( n =7) thymi. Values shown are relative to one wild-type animal. (C) ChIP-qPCR for FLAG-PRDM14 at the putative PRDM14 binding site in Notch1 intron 4, a non-specific intronic site 2.5 kb downstream of the putative Notch1 binding site, and a gene desert located on Chr 6 that contains no genes (Neg Ctrl) in unsorted whole bone marrow of wild-type ( n =3) and R26FLPR;Mx1-cre ( n =3) mice at 10 days post-pIpC injection. (D) ChIP-qPCR for H3K4me3 at the Notch1 5′ and 3′ cRSSs in wild-type ( n =5) and R26PR;Mx1-cre ( n =5) thymi. Mean±s.d. shown for all graphs. * P ≤0.05, ** P ≤0.01.

Article Snippet: Antibodies were used at manufacturer-recommended concentrations from Active Motif: H3K4me1 (39297), H3K4me3 (39915), H3K9me2 (39753), H3K27me3 (39155), H3 (61277), Cell Signaling Technology: IKAROS (5443), Cleaved NOTCH1 (Val1744) (4147), Sigma-Aldrich: FLAG M2 (F1804), Bio-Rad: HRP-goat-anti-mouse, and Jackson Immunoresearch: HRP-goat-anti-rabbit.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, ChIP-qPCR, Injection

Journal: PLoS Genetics

Article Title: SCL , LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

doi: 10.1371/journal.pgen.1004768

Figure Lengend Snippet: ( A ) Analysis of SCL-LMO1-upregulated genes in Cd3ε -/- thymocytes. Gene signatures were analysed using the Stem Cell Discovery Engine tool as described in Experimental procedures, and signatures deemed enriched in SCL-LMO1 up-regulated genes (adjusted p-val <0.05) were classified into broad categories. The heatmap depicts the frequency of association to each gene by signature categories (stem cells, cancer, and other). ( B ) GSEA analysis of hematopoietic transcription factor signatures in SCL-LMO1 thymocytes. The lists of genes bound by 31 hematopoietic transcription factors within 2 kb of their proximal promoters were extracted from a compendium of ChIP-seq experiments (see ). The top 7 transcription factors are illustrated (FDR, false discovery rate, ranging from 0.01–0.32). In comparison, NOTCH1-bound genes were not up- or down-regulated by SCL-LMO1 . ( C ) Hierarchical organisation of the self-renewal network controlled by SCL-LMO1. Integration of published ChIP-seq data with up-regulated genes in DN3 pre-leukemic thymocytes identified common targets of SCL, LMO2 and LYL1 (highlighted in yellow). Incoming edges represent the binding of regulators at the proximal promoters of target genes (peaks within 2kb of the transcription initiation sites). ( D ) SCL and LMO1 occupy Lyl1 regulatory sequences. Chromatin extracts from the AD10.1 DN cell line expressing SCL (+SCL) or not (-SCL) were immunoprecipitated with the indicated antibodies. Lyl1 regulatory sequences were amplified by q-PCR. Data are expressed as fold enrichment over IgG controls. ( E ) Lyl1 gene expression is induced by SCL-LMO1 but is not modified by Notch1 in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to β-Actin (Mean +/- SD, n = 3).

Article Snippet: Transgenic mice were previously described: pSil-TSCL ( SCL tg ) , Lck - LMO1 ( LMO1 tg ) and Lck - NotchIC9 ( Notch1 tg ) (NIAID/Taconic Repository Bethesda), E2a +/- , Lck - LYL1 ( LYL1 tg ) (International Mouse Strain Resource), Transgenic Notch Reporter ( TNR tg ) (Tg(Cp-EGFP)25Gaia, The Jackson Laboratory, Maine, United States) and Cd3ε -/- .

Techniques: ChIP-sequencing, Comparison, Binding Assay, Expressing, Immunoprecipitation, Amplification, Gene Expression, Modification, Purification, Transgenic Assay, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: SCL , LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

doi: 10.1371/journal.pgen.1004768

Figure Lengend Snippet: ( A ) Generation of transgenic mice expressing the LMO1-binding defective mutant SCLm13. The sequence coding for wild type human SCL or human SCLm13 HLH domain mutant were cloned into the VA h CD2 cassette to generate transgenic mice. Shown are amino acids of the HLH region of SCL or SCLm13. ( B ) Immunofluorescence of human SCL (wt or m13) by flow cytometry. Thymocytes were stained with the monoclonal antibody against human SCL (BTL73). Control cells were stained with the second antibody only. ( C ) Expression of E protein target genes is inhibited both by SCL-LMO1 and SCLm13-LMO1 transgenes in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to β-Actin (Mean +/- SD, n = 3). ( D ) Kaplan-Meier curves of the time to leukemia for LMO1 tg , E2a +/- LMO1 tg , SCL tg LMO1 tg and SCLm13 tg LMO1 tg mice. ( E ) The interaction between SCL and LMO1 is required to activate the transcription of the self-renewal genes Lyl1 , Hhex and Nfe2 in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to β-Actin (Mean +/- SD, n = 3). ( F–G ) SCL but not the LMO1-binding defective SCL-m13 mutant collaborates with LMO1 to induce abnormal thymic reconstitution potential to thymocytes. Pre-leukemic thymocytes (1.5×10 7 cells) from 3-week-old mice were transplanted. Recipient mice were analysed for thymic reconstitution (CD45.2 + Thy1 + ) after 6 weeks (F) and the proportion of DP cells in engrafted CD45.2 + Thy1 + thymocytes was assessed by FACS (G).

Article Snippet: Transgenic mice were previously described: pSil-TSCL ( SCL tg ) , Lck - LMO1 ( LMO1 tg ) and Lck - NotchIC9 ( Notch1 tg ) (NIAID/Taconic Repository Bethesda), E2a +/- , Lck - LYL1 ( LYL1 tg ) (International Mouse Strain Resource), Transgenic Notch Reporter ( TNR tg ) (Tg(Cp-EGFP)25Gaia, The Jackson Laboratory, Maine, United States) and Cd3ε -/- .

Techniques: Transgenic Assay, Expressing, Binding Assay, Mutagenesis, Sequencing, Clone Assay, Immunofluorescence, Flow Cytometry, Staining, Control, Purification, Quantitative RT-PCR