cf r d systems 1277 jg recombinant human jagged 2 fc chimera protein  (R&D Systems)

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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: 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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: 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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: 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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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: 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: For NOTCH activation experiments, tissue culture wells were pre-coated overnight at room temperature (RT) with Protein G (Prospec, East Brunswick, NJ) at 50 ug/ml, washed twice in phosphate buffered saline (PBS), blocked in 1% BSA/PBS for 2 hours at RT, washed three times with PBS, coated with either human recombinant chimeric Jag1 fused to an FC fragment (R&D Systems, Minneapolis, MN) or control purified IgG FC protein (Jackson ImmunoResearch, West Grove, PA) at 2 ug/ml in 0.1% BSA/PBS for 3 hours RT, stored overnight at 4 0 C, and washed three times immediately before use.

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