Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: A) The purity of isolated cells was tested using anti-Notch1 antibody (green). Propidium iodide (PI, red) was used to visualize the nucleus of the cell. Bar is 50 μm. B) The levels of Notch1 expression in isolated cells were higher compared to expression of Notch1 in whole retinas. Gene expression was measured by qRT-PCR. Results are expressed as a percentage of the corresponding value in the Notch1 + cells isolated from embryonic day 14 developing retinas ± SEM (*P < 0.05).

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Isolation, Expressing, Gene Expression, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: Functional annotation for the selected genes revealed by One-Way ANOVA.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Functional Assay

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: For each gene, results are expressed as percentages ± SEM of the corresponding values in the Notch1 + cells isolated from E14 developing retinas (*P < 0.05).

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: The expression of retinal cell markers in Notch1 + progenitor cells and in whole retina samples at E14 and P0.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: The putative members of E14 Notch1 and P0 Notch1 gene networks.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques:

Journal: PLoS ONE

Article Title: Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

doi: 10.1371/journal.pone.0131054

Figure Lengend Snippet: List of RT-PCR primers.

Article Snippet: Cells were then incubated with Notch1 primary antibody (130-101-868, Miltenyi Biotec Inc., San Diego, CA) followed by species-specific secondary fluorescent antibodies (Invitrogen, Carlsbad, CA).

Techniques:

Journal: Stem Cells (Dayton, Ohio)

Article Title: Thyroid Hormone‐Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis

doi: 10.1002/stem.2544

Figure Lengend Snippet: Expression profiles of genes involved in Notch signaling in X. laevis intestine during natural and T3‐induced metamorphosis. Quantitative real‐time RT‐PCR was performed using total RNAs prepared from the intestine of animals at indicated developmental stages ( A, C, E, G, I, K, M) or stage‐54 tadpoles after 10 nM T3 treatment ( B, D, F, H, J, L, N ). Levels of Hairy1 ( A, B ), Hairy2b ( C, D ), DLL1 ( E, F ), DLL3 ( G, H ), Jag1 ( I, J ), Jag2 ( K, L ), and Notch1 ( M, N ) mRNAs are shown relative to those of ribosomal protein L8 (rpL8) mRNA, with the values at stage 54 or 0‐day treatment set to 1. Error bars represent the SEM ( n = 7 for A–D ; n = 3 for E–H , J, M, N ; n = 8 for I, L ; n = 5 for K ). The values were analyzed by ANOVA followed by Scheffe's post hoc test whose results are shown only for the adjacent stages or days except for Notch1 ( M ). Asterisks indicate that the mRNA levels are significantly different. *, p < .05, **, p < .01; ns: not significant.

Article Snippet: The sections of the intestine from stage‐62 tadpoles were double‐immunostained with a mixture of the mouse anti‐human cytokeratin 19 (CK19) (1:100; Novocastra), which is a predominant cytokeratin in the adult Ep including the stem cells , and the rabbit anti‐human DLL1 (1:100; Abcam, Tokyo, Japan), the rabbit anti‐human Jag1 (1:50; Santa Cruz Biotechnology, Dallas, TX), or the rabbit anti‐human Notch1 (1:500; Rockland Inc., Gilbertsville, PA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Stem Cells (Dayton, Ohio)

Article Title: Thyroid Hormone‐Induced Activation of Notch Signaling is Required for Adult Intestinal Stem Cell Development During Xenopus Laevis Metamorphosis

doi: 10.1002/stem.2544

Figure Lengend Snippet: Correlation of the expression patterns of Notch pathway components with that of a stem/progenitor marker in the metamorphosing intestine. Cross‐sections of the intestine from tadpoles at stages 60 ( A, D ) and 61 ( B, E, G, I ), and stage‐54 tadpoles treated with 10 nM T3 for 3 ( C, F ) and 5 days ( H, J ) were hybridized with antisense Hairy1 ( A‐C ) or Hairy2b ( G, H ) probes. The corresponding serial sections were hybridized with antisense LGR5 probe ( D–F, I, J ) for comparison. Hairy1 is expressed in the adult epithelial stem (AE)/progenitor cells (A, arrowhead) when they first appear as the small islets expressing LGR5 ( D , arrow) at stage 60. These islets grew in size as metamorphosis proceeded and continued to co‐express Hairy1 ( B , arrowheads) and LGR5 ( E , arrows). During T3‐induced metamorphosis, Hairy1 ( C , arrowhead) is also expressed in the AE/progenitor cells expressing LGR5 ( F , arrow) after 3 days of T3 treatment. Some, although not all, cells expressing Hairy2b were in the connective tissue (CT) ( G , arrowheads) underlying the AE/progenitor cells expressing LGR5 ( I , arrows). Hairy2b is also expressed in the CT after 5 days of T3 treatment ( H , arrowheads), again, with some underlying the AE/progenitor cells ( J , arrows). Cross‐sections of the intestine from tadpoles at stage 62 were double‐immunostained with anti‐CK19 to detect the islet of AE/progenitor cells ( K–M , red) and anti‐DLL1 ( K , green), anti‐Jag1 ( L , green), or anti‐Notch1 ( M , green) followed by counterstaining with DAPI ( K–M , blue). DLL1 ( K , arrowheads) and Notch1 ( M , arrowheads) are expressed in the islets (arrows). The cells expressing Jag1 are scattered in both the larval Ep and CT with some just beneath the islet, but not in the islets ( L , arrowheads). The dashed‐lines indicate the boundary of the Ep and the CT. Scale bars = 20 μm. Abbreviations: AE, adult epithelial stem/progenitor cells, CT, connective tissue, Ep, epithelium.

Article Snippet: The sections of the intestine from stage‐62 tadpoles were double‐immunostained with a mixture of the mouse anti‐human cytokeratin 19 (CK19) (1:100; Novocastra), which is a predominant cytokeratin in the adult Ep including the stem cells , and the rabbit anti‐human DLL1 (1:100; Abcam, Tokyo, Japan), the rabbit anti‐human Jag1 (1:50; Santa Cruz Biotechnology, Dallas, TX), or the rabbit anti‐human Notch1 (1:500; Rockland Inc., Gilbertsville, PA).

Techniques: Expressing, Marker, Comparison

Journal: Nutrients

Article Title: Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

doi: 10.3390/nu8070439

Figure Lengend Snippet: Primers for reverse transcriptase polymerase chain reaction RT-PCR.

Article Snippet: The membranes were subsequently blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and were incubated with primary antibodies against cytokeratin 20 (CK20, Abcam, Cambridge, MA, USA), Notch1 (Novus Biologicals, Littletown, CO, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-GSK3β (Cell Signaling Technology, Danvers, MA, USA) overnight.

Techniques: Reverse Transcription, Polymerase Chain Reaction

Journal: Nutrients

Article Title: Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

doi: 10.3390/nu8070439

Figure Lengend Snippet: WPE and its bioactive compounds suppress colon CSCs markers, including CD133, CD44, DLK1, and Notch1 as well as Wnt/β-catenin signaling in colon CSCs. CD133 + CD44 + HCT116 cells were treated with varying concentrations of WPE (0, 10, 20, and 40 μg/mL) ( A a , Ba ); or concentrations of (+)-catechin, chlorogenic acid, ellagic acid and gallic acid comparable to 40 μg/mL of WPE ( A b , Bb ) for 6 days. mRNA expressions of CD133, CD44, DLK1 and Notch1 were examined by RT-PCR, and β-actin was used as a loading control ( A ); Protein levels of Notch1, β-catenin and p-GSK3β were examined by Western blot analysis, and α-tubulin was used as a loading control ( B ). Representative blots are shown in left panel and quantified in right panel. The values shown are the means ± SEM. A p -value < 0.05 was considered significant. Ctrl, Control; WPE, walnut phenolic extract; CSCs, cancer stem cells; CAT, (+)-catechin; CGA, chlorogenic acid; EA, ellagic acid; GA, gallic acid.

Article Snippet: The membranes were subsequently blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and were incubated with primary antibodies against cytokeratin 20 (CK20, Abcam, Cambridge, MA, USA), Notch1 (Novus Biologicals, Littletown, CO, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-GSK3β (Cell Signaling Technology, Danvers, MA, USA) overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Nutrients

Article Title: Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

doi: 10.3390/nu8070439

Figure Lengend Snippet: WPE down-regulates CD133, CD44, DLK1, and Notch1 in human primary cells from colorectal cancer tissue. Primary cancer cells were treated with varying concentrations of WPE (0, 10, 20, and 40 μg/mL) for 3 d. mRNA expressions of CD133, CD44, DLK1 and Notch1 were detected by RT-PCR, and GAPDH was used as a loading control. The values shown are the means ± SEM. A p -value < 0.05 was considered significant. Ctrl, Control.

Article Snippet: The membranes were subsequently blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and were incubated with primary antibodies against cytokeratin 20 (CK20, Abcam, Cambridge, MA, USA), Notch1 (Novus Biologicals, Littletown, CO, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-GSK3β (Cell Signaling Technology, Danvers, MA, USA) overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Cartoon schematic of the design strategy for the DLL4 Site 3 mutant library. Red spheres depict mutated interface residues. ( B ) Table depicting DLL4 interface residue positions and the mutations allowed at each position and schematic of DLL4 yeast display construct. Yellow stars indicate mutations. ( C ) Flow cytometry histogram plots of yeast stained with fluorescently-labeled Notch3 protein following each round of selection (left), and a table indicating the frequency of mutations and consensus DLL4 mutant sequence (right). ( D ) Structural models highlighting the mutated residues in DLL4.v2. WT residues in DLL4 and mutated residues in DLL4.v2 are colored orange and shown in surface representation. DLL4 models were based on DLL4:Notch1 (PDB ID: 4XL1, for C2-DSL-EGF1 domains) and Jagged1:Notch1 (PDB ID:5UK5, for EGF1-EGF3) structures.

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Mutagenesis, Residue, Construct, Flow Cytometry, Staining, Labeling, Selection, Sequencing

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: Cartoon representation showing the structural context of DLL4.v2 mutants in a model of the rat Jag1-Notch1 complex (PDB ID:5UK5). ( A ), ( B ), ( C ), ( D ), and ( E ) are zoom panels depicting the residues that surround the mutated position. Numbers and dashes in (E) are inter-atomic distances atoms measured in angstroms.

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques:

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Schematic depicting the generation of DLL4 MAX through engraftment of DLL4.v1 and DLL4.v2 mutations. Red spheres indicate affinity-enhancing mutations. ( B ) SPR binding isotherms measuring DLL4 variant interactions with the ligand binding domains of Notch1-4. The table shows the K D determined for each interaction and the fold-enhancement of DLL4 MAX affinity relative to WT DLL4. ( C ) Representative SPR sensograms depicting the binding of 200 nM concentrations of WT DLL4, DLL4.v1, DLL4.v2, and DLL4 MAX to Notch1 EGF6-13. ( D ) DSF was used to determine the T m of WT DLL4 and Delta MAX .

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Binding Assay, Variant Assay, Ligand Binding Assay

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: (A-B) SEC chromatograms from the purification of the ligand-binding regions of human Notch1-4 (A) and murine Notch1 (B) . All proteins were purified by Ni-NTA followed by size exclusion chromatography using a Superdex S75 column. (C) SDS-PAGE gels showing the purity and molecular weight of each Notch construct. ( D ) SEC chromatograms from the purification of DLL4, DLL4.v1, DLL4.v2, and Delta MAX proteins. All proteins were purified by Ni-NTA followed by size exclusion chromatography using a Superdex S200 column. (E) SEC chromatograms from the purification of C-termini biotinylated WT DLL4 and Delta MAX proteins. (F) SDS-PAGE gels showing the purity and molecular weight of each DLL4 construct. (G) SEC chromatograms from 1L protein preps were overlaid to highlight the increased yield of recombinant Delta MAX compared to WT DLL4.

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Purification, Ligand Binding Assay, Size-exclusion Chromatography, SDS Page, Molecular Weight, Construct, Recombinant

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) DLL4 variants were non-specifically adsorbed to 96-well tissue culture plates or immobilized to streptavidin plates. Next, CHO-K1 Notch1-Gal4 cells were added to plates and Notch activation was measured by flow cytometry. The gating strategy to quantify Notch1 activation based on expression of H2B-mCitrine using CHO reporter cells is detailed. HEK293T were transduced to generate stable cell lines expressing WT DLL4 or Delta MAX and sorted to normalize the expression of DLL4. ( B ) Expression of WT DLL4 and Delta MAX on HEK293T was analyzed with anti-hDLL4 PE antibody using flow cytometry. The sorted cell lines were used for Notch activation assays in co-culture with Notch1, Notch2, and Notch3-U2OS luciferase reporters.

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Activation Assay, Flow Cytometry, Expressing, Stable Transfection, Co-Culture Assay, Luciferase

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Dose-titration assay comparing the Notch1 reporter activity stimulated by WT DLL4 and Delta MAX proteins that were non-specifically adsorbed to tissue culture plates. ( B ) Dose-titration assay comparing the Notch1 reporter activity stimulated by WT DLL4 and Delta MAX proteins. DLL4 ligands were C-termini biotinylated and coupled to streptavidin-coated plates. ( C ) Time-course experiment comparing Notch1 reporter activity stimulated by WT DLL4 or Delta MAX immobilized at 50 nM. ( D ) A luciferase reporter assay was used to measure Notch1, Notch2, or Notch3 activation by WT DLL4 and DLL4 MAX . Protein concentrations were a 10-fold serial dilution from 50 nM to 0.05 nM. ( E ) Detection of endogenous cleaved Notch intracellular domain (N1ICD) in U2OS and MCF-7 cell lines by Western blot. Cells were Notch activated with 50 nM of WT DLL4 and Delta MAX immobilized in streptavidin plates. ( F ) Co-culture assay comparing Notch1 activation by Delta MAX and WT DLL4. Notch1 reporter cells and ligand-expressing cells were cultured at a 1:1 ratio. ( G ) Notch1 luciferase reporter cells were stimulated with Delta MAX protein in different formats. Yeast expressing Delta MAX were co-cultured in 10:1 ratio with reporter cells; HEK293T cells stably expressing Delta MAX were co-cultured in 1:1 ratio; Delta MAX -coated streptavidin beads were mixed in 20:1 ratio; 100 nM Delta MAX was non-specifically adsorbed to surfaces prior to addition of reporter cells; 10 nM C-terminally biotinylated Delta MAX was immobilized on streptavidin-coated plates. Notch activation was normalized to the corresponding controls. Delta MAX statistics are referred to WT DLL4. *P<0.05, **P<0.01, ****P<0.0001, ns: not significant (Two-way ANOVA).

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Titration, Activity Assay, Luciferase, Reporter Assay, Activation Assay, Serial Dilution, Western Blot, Co-culture Assay, Expressing, Cell Culture, Stable Transfection

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Cartoon representation of different ligand-presentation formats. ( B ) Yeast expressing Delta MAX (N-EGF5) were co-cultured with CHO-K1 reporter cells for 24h at different ratios and fluorescence was measured by flow cytometry. ( C ) HEK293T cells expressing Delta MAX were co-cultured with Notch1 reporter cells at various ratios for 24h and Notch activation was measured by flow cytometry. ( D ) Magnetic beads were pre-coated with biotinylated Delta MAX and co-cultured with CHO K1 reporter cells at ratios of 1:20 or 1:40 to stimulate Notch activation. ( E ) Flow cytometry dot plots depict the expression level of Notch1 and Delta MAX .

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Expressing, Cell Culture, Fluorescence, Flow Cytometry, Activation Assay, Magnetic Beads

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Dose-titration assay measuring the inhibition of Notch1 reporter activity by soluble DLL4 variants. Notch1 signaling was activated by culturing cells on streptavidin plates coated with 50 nM WT DLL4. ( B ) Dose-titration assay comparing the inhibition potency of DAPT, BB-94, and soluble Delta MAX . Notch1 signaling was activated by culturing cells on streptavidin plates coated with 50 nM WT DLL4. ( C ) Soluble WT DLL4 and Delta MAX at 3 µM were tested for their ability to inhibit Notch1 activation by 293T cells overexpressing DLL4, DLL1, JAG1, or JAG2. ( D ) Dose-titration assay comparing the ability of WT DLL4 and Delta MAX to inhibit activation of Notch1-3 by WT DLL4. 10-fold serial dilutions starting at 3,000 nM of soluble DLL4 or Delta MAX were added to Notch1, Notch2, and Notch3 luciferase reporter cells. Notch signaling was activated by culturing cells on streptavidin plates coated with 50 nM WT DLL4. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant (Two-way ANOVA).

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Titration, Inhibition, Activity Assay, Activation Assay, Luciferase

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Dose-titration assay comparing the inhibition potency of DAPT and Delta MAX . Fluorescent Notch1 reporter cells were cultured in a 1:1 ratio with HEK293T cells stably expressing WT DLL4. ( B ) Summary of the gating strategy used for flow cytometry to differentiate between HEK293T and CHO cell signals. The basal expression of H2B-mCitrine in CHO cells was used as a criterion to distinguish between cell types. Notably, the population identified in the middle and bottom panels (Notch1 reporter CHO cells) corresponds to approximately 50% of the total cells, which is consistent with a 1:1 ratio of CHO cells to 293T cells.

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Titration, Inhibition, Cell Culture, Stable Transfection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

doi: 10.1101/2022.03.07.483330

Figure Lengend Snippet: ( A ) Human PBMCs CD8 + T cells were co-cultured with K32 artificial antigen-presenting cells (aAPCs) engineered to express WT DLL4 or Delta MAX for simultaneous Notch and TCR stimulation. ( B ) aAPCs were assayed for expression of WT DLL4 or Delta MAX using flow cytometry. K32 cells were loaded with OKT3 antibody using a gradient of concentrations starting at 0.0125 µg/ml, and then co-cultured with CD8 + T cells in ratio 10:1. After 96h, CD8 + T cells were analyzed for proliferation ( C ) and IFNγ secretion ( D ). (E) T cells were stimulated for 24, 48, and 96h to quantify mRNA levels of intracellular activation markers by real-time PCR, including IFNγ, Granzyme B and Hes-4. ( F ) K32 cells expressing WT DLL4 or Delta MAX were co-culture with human CD8 + T cells (ratio 1:10) for detection of Notch activation by Western blot. Co-cultured CD8 + T cells were positively sorted by MACS column after cultivation with K32 cells, and nuclear extracts were harvested after 96 hours. Cleavage of Notch1 and Notch2 were examined by Western blot with specific antibodies. β-actin was used for loading control. Western blot images are representative results of 3 biological repeats. NA: non-activated T cells. Statistics for Delta MAX are referred to WT DLL4. *P<0.01, **P<0.001, ***P<0.001, ***P<0.0001, ns: not significant difference (Two-way ANOVA). T cell proliferation and IFNγ secretion data were analyzed by unpaired t tests.

Article Snippet: Antibodies used in this study were: anti-hNotch1 PE (Biolegend), anti-hNotch2 AF647 (Biolegend), anti-hNotch3 PE (Biolegend), anti-hDLL4 PE (Biolegend), anti-hDLL1 PE (Biolegend), anti-hJagged1 AF647 (Biolegend), anti-hJagged2 PE (Biolegend), and anti-HA tag AF488 (Cell Signaling Technologies), anti-hCD8a Bv785 (Biolegend), anti-hIFNγ APC (Biolegend), goat anti-mouse IgG (secondary antibody, KPL), goat anti-rabbit IgG (secondary antibody, Vector laboratories), anti-cleaved Notch1 (Val1744, Cell Signaling Technology), anti-Notch2 (D76A6, Cell Signaling Technologies), anti-β-Actin (Cell Signaling Technology and Sigma).

Techniques: Cell Culture, Expressing, Flow Cytometry, Activation Assay, Real-time Polymerase Chain Reaction, Co-Culture Assay, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Notch was found up-regulated after azelaic acid (AZA) treatment. (A) 120 differential expressed proteins associated in 52 functions after AZA treatment by protein mass spectrometry analysis in the heat map. (B) GO annotation with 528 differential expressed proteins. (C, D) THP-1, Molm-13, NK, and T cells were treated with 10 µM AZA for 24 h, the expression level of ICN1 and ICN2 was validated by western blot. (E) The expression of Notch1 and Notch2 in mouse spleen measured by ICH. IHC, immunohistochemistry.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Mass Spectrometry, Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway

doi: 10.3389/fphar.2019.01396

Figure Lengend Snippet: Azelaic acid (AZA) exerts anti-leukemic effect by activating the Notch signaling pathway. (A) Notch responsive elements were transfected into 293T cells after 24 h. Cells were then treated with 10 µM AZA, 10 µM RO4929097, and combination for 24 h, the Notch signaling reporter assay was measured by dual luciferase reporter activity. (B) Validation of the RNA expression of Notch1 and Notch2, the downstream target genes HES1 and HEY1 in Molm-13 and THP-1 cells by qPCR. (C) The protein expression level of ICN1, ICN2, HEY1, and HES1 in acute myeloid leukemia (AML) cell lines after treatment of AZA and RO4929097 and their detection by western blot. ImageJ was used for the densitometric analysis. Data represent means ± SD. (D) Molm-13 cells were pretreated with 10 µM RO4939097 for 24 h, and then treated with 10 µM AZA for another 24 h. Cells were collected for apoptosis analysis. (E) NK cells and T cells were pretreated with 10 µM AZA, 10 µM RO4929097, and combination for 24h before co-culture with THP-1 cell and Molm-13 cells at an E:T ratio of 5:1. The cytotoxicity of NK and T was determined by detecting the LDH release rate. (F) NK and T cells were pre-treated with AZA and RO4929097, then co-cultured with THP-1 cell at an E:T ratio of 3:1 for 4 h. The level of TNF-α and IFN-γ in the supernatant was measured by ELISA. A Total of three independent experiments were performed. *P < 0.05, **P < 0.01,***P < 0.001.

Article Snippet: The following antibodies were used: CD3(Cat# 100203), CD4 (Cat# 100431), CD8 (Cat# 100761), CD107a (Cat# 328605), TRAIL (Cat# 308205), CD25 (Cat# 302605), and CD69 (Cat# 310903) were purchased from BioLegend (USA), ICN1 (Cat# CSB-PA084572), ICN2 (Cat# CSB-PA964902) were from CUSABIO (USA); GAPDH (Cat# 6004-1), β-actin (Cat# 14395-1) were from ProteinTech (USA).

Techniques: Transfection, Reporter Assay, Luciferase, Activity Assay, Biomarker Discovery, RNA Expression, Expressing, Western Blot, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay