Journal: STAR Protocols

Article Title: Protocol to use TopNet for gene regulatory network modeling using gene expression data from perturbation experiments

doi: 10.1016/j.xpro.2022.101737

Figure Lengend Snippet:

Article Snippet: TaqMan probe detecting murine Jagged 2 (Jag2) , Applied Biosystems , Mm00439935_m1.

Techniques: Virus, Retroviral, Plasmid Preparation, Recombinant, Derivative Assay, Reverse Transcription, SYBR Green Assay, Binding Assay, Transgenic Assay, Expressing, Construct, Sequencing, Software

Journal: Nucleic Acids Research

Article Title: Regulation of alternative polyadenylation in the yeast Saccharomyces cerevisiae by histone H3K4 and H3K36 methyltransferases

doi: 10.1093/nar/gkaa292

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: RNAP II CTD Ser2-P , ChromoTek , 3E10.

Techniques:

Journal: Nucleic Acids Research

Article Title: Regulation of alternative polyadenylation in the yeast Saccharomyces cerevisiae by histone H3K4 and H3K36 methyltransferases

doi: 10.1093/nar/gkaa292

Figure Lengend Snippet: Summary of key results

Article Snippet: RNAP II CTD Ser2-P , ChromoTek , 3E10.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: NOTCH ligands JAG1 and JAG2 as critical pro-survival factors in childhood medulloblastoma

doi: 10.1186/2051-5960-2-39

Figure Lengend Snippet: Expression of NOTCH ligands in MB primary tumors. (a) Relative mRNA expression of the indicated NOTCH ligands in a representative gene expression dataset of 62 human MB tumors . Dot plots showing relative expression of JAG1 (b) , JAG2 (c) , DLL1 (d) , DLL3 (e) , and DLL4 (f) in three independent gene expression datasets of human MB tumors: 62 samples ; 57 samples ; 76 samples .

Article Snippet: Probe-primer solutions specific for the following genes (purchased from Applied Biosystems) were used: MYC (Hs00153408_m1), JAG2 (Hs00171432_m1), JAG1 (Hs01070032_m1), and HES1 (Hs00172878_m1).

Techniques: Expressing, Gene Expression

Journal: Acta Neuropathologica Communications

Article Title: NOTCH ligands JAG1 and JAG2 as critical pro-survival factors in childhood medulloblastoma

doi: 10.1186/2051-5960-2-39

Figure Lengend Snippet: Expression of NOTCH ligands across MB molecular subgroups and MYC / JAG2 expression correlation in MB primary tumors , in histological MB subtypes , and in distinct metastatic MB stages. Dot plots showing relative expression (log2) of JAG1 (a) , JAG2 (b) , DLL1 (c) , and DLL3 (d) , across MB subgroups in a representative dataset of 103 human MB tumors : SHH, n = 33; WNT, n = 8; Grp3 (Group 3), n = 27; Grp4 (Group 4), n = 35. (e) Correlation between MYC and JAG2 mRNA expression (log2) in a representative dataset of 103 human MB tumors . r: Pearson’s value; p: p values. (f) Correlation study of JAG2 and MYC levels in Group 3 MB cases . Box plots showing JAG2 (g) and MYC (h) expression (log 2) according to the following MB histological variants : classic (n = 51), desmoplastic (desmop.) (n = 6), and (LCA) large cells/anaplastic (n = 17); center line = median. Box plots showing JAG2 (i) and MYC (j) expression (log 2) in MB tumors clustered according to metastatic stage : M0, n = 42; M1, n = 7; ≥M2, n = 9; center line = median.

Article Snippet: Probe-primer solutions specific for the following genes (purchased from Applied Biosystems) were used: MYC (Hs00153408_m1), JAG2 (Hs00171432_m1), JAG1 (Hs01070032_m1), and HES1 (Hs00172878_m1).

Techniques: Expressing

Journal: Acta Neuropathologica Communications

Article Title: NOTCH ligands JAG1 and JAG2 as critical pro-survival factors in childhood medulloblastoma

doi: 10.1186/2051-5960-2-39

Figure Lengend Snippet: MYC - dependent regulation of NOTCH ligand JAG2 expression. (a) Relative mRNA expression (upper panel) of MYC (black bars, left Y axis) and JAG2 (orange bars, right Y axis) in the indicated cell lines. Values represent the fold-change in mRNA expression relative to cerebellum (defined as 1). Protein expression (lower panel) of MYC and JAG2 in the corresponding cell lines. (b) Relative mRNA expression (upper panel) of MYC and JAG2 in DAOY cells and stable clones of DAOY V11, and DAOY M2.1 cells. Values represent fold-change in mRNA expression relative to cerebellum (defined as 1). Protein expression (lower panel) of MYC and JAG2 in corresponding cells is shown. (c) MYC and JAG2 mRNA (upper panel) and protein (lower panel) expression in DAOY M2.1 cells transfected with MYC siRNA compared to control siRNA. mRNA values represent the percent decrease in JAG2 and MYC expression relative to control. Expression of β-actin was used as a control for western blot analysis. Statistical analysis: black stars indicate p values relative to MYC expression; orange stars indicate p values relative to JAG2 expression. (d) ChIP-on-chip data show occupancy of the JAG2 genomic sequence by MYC in four MB cell lines (from top to bottom: DAOY, UW-228, D458, and D341). (e) Time-dependent caspase 3/7 activation in DAOY M2.1 cells upon JAG2 siRNA treatment at the indicated time-points; values represent the percent increase in caspase 3/7 activity relative to control. (f) Proliferation status of DAOY cells, DAOY V11, DAOY M2.1, and DAOY M2.1 cells at 48 hours after JAG2 siRNA treatment compared to control siRNA. Caspase 3/7 activation at 48 hours after treatment with GSI in the indicated cell lines (g) and DAOY-derived clones (h) . Low MYC cells: DAOY; high MYC cells: DAOY M2.1. Values represent the percent increase in caspase 3/7 activity relative to control siRNA.

Article Snippet: Probe-primer solutions specific for the following genes (purchased from Applied Biosystems) were used: MYC (Hs00153408_m1), JAG2 (Hs00171432_m1), JAG1 (Hs01070032_m1), and HES1 (Hs00172878_m1).

Techniques: Expressing, Clone Assay, Transfection, Control, Western Blot, Sequencing, Activation Assay, Activity Assay, Derivative Assay

Journal: Acta Neuropathologica Communications

Article Title: NOTCH ligands JAG1 and JAG2 as critical pro-survival factors in childhood medulloblastoma

doi: 10.1186/2051-5960-2-39

Figure Lengend Snippet: MYC - dependent NOTCH signaling in MB cells. Simplified scheme illustrating the MYC-dependent NOTCH molecular network involving JAG1 and JAG2 in MB cells. (Left panel) Low MYC MB cell. (Right panel) High MYC MB cell.

Article Snippet: Probe-primer solutions specific for the following genes (purchased from Applied Biosystems) were used: MYC (Hs00153408_m1), JAG2 (Hs00171432_m1), JAG1 (Hs01070032_m1), and HES1 (Hs00172878_m1).

Techniques:

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Heatmap for gene expression by RNA-seq reveals active Notch signaling in freshly isolated MuECs and MuSCs, but not in myofibers . (B) Upper panel: Schematic genomic structure of Jag2 LoxP/LoxP locus for conditional Jag2 mutant ( LacZ/Neo-Jag2 LoxP/LoxP ) mice before Flippase (Flp)-mediated recombination. Middle panels: On X-gal staining, capillaries (white arrow) in TA muscle from LacZ/Neo-Jag2 LoxP/LoxP mice are positive for LacZ(+) and CD31. Lower panels: MuSCs from LacZ/Neo-Jag2LoxP/LoxP mice are positive for LacZ(+) and Pax7 or MyoD. (C) qRT-PCR shows upregulation and downregulation of Dll1 and Jag2 following MuSC activation and differentiation, respectively. P0, P3, P5, Diff 1, and Diff 3 denote freshly isolated MuSCs, passage day 3, passage day 5, differentiation day 1, and differentiation day 5. (D) Anti-Jag2 antibody staining shows Jag2 expression at the membrane of MuECs and MuSCs. DAPI stained all nuclei (blue). Scale

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Gene Expression, RNA Sequencing, Isolation, Mutagenesis, Staining, Quantitative RT-PCR, Activation Assay, Expressing, Membrane

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Muscle from 4 day-old and 3-month-old Jag2 sm homozygous mice showed (B) reduced fiber diameters, (C) Pax7(+) MuSCs (arrows), and (D) increased Sirius red (+) fibrosis vs. WT mice. (E) Grip strength is reduced in Jag2 sm homozygous vs. WT mice. (F, G) Treadmill running time and distance are reduced in Jag2 sm homozygous vs. WT mice. (H) Motor coordination or balance on the rotarod was impaired in Jag2 sm homozygous vs. WT mice. DAPI stained all nuclei (blue). Scale bars, 100 μ m. An unpaired t-test showed *, p<0.05; **, p <0.01. ***, p <0.001. Error bars show SEM.

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Staining

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) WT: Pax7 tdT and Jag2 sm :Pax7 tdT mice were injected with TMX prior to sacrifice. (B, C, D) TA muscle sections and isolated single muscle fibers demonstrated reduced Pax7(+) MuSCs (arrows) in Jag2 sm homozygous vs. WT mice. Scale bars; 20 µ m (top panels), 50 µ m (fibers). (E, F) Freshly isolated MuSCs from homozygous Jag2 sm mice show reduced colony sizes and EdU(+) proliferating cells. (G) MuSCs isolated from homozygous Jag2 sm mice show (H) reduced EdU(+) proliferating cells (green), (I) MyHC(+) myogenic differentiation (green) and fusion in days 1 and 3 differentiation conditions, while (J) MyoD(+) cells (red) are not altered, compared with WT cells. DAPI stained all nuclei (blue). An unpaired t-test showed **, p <0.01, ***, p<0.001. Error bars show SEM.

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Injection, Isolation, Staining

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Single or repeated CTX injections into the TA muscle were performed on WT and Jag2 sm mice. (B) TA histology [hematoxylin & eosin (H&E), Oil red-O] and immunostaining (eMyHC/Laminin/DAPI and CD31/Laminin) 7 days following CTX injection into the TA. Scale bars from left to right: 100, 25, 50 and 250μm. (C) H&E staining 21+7 days following sequential CTX injections into the TA. Scale bar, 100 μm. Feret’s diameters of TA fibers in WT and Jag2 sm homozygous mice at (D) 7 days and (E) following CTX injection. (F) Oil red-O (+) area was evaluated at 7 days following CTX injection. (G) Feret’s diameters of TA myofibers in WT and Jag2 sm homozygous mice 21+7 days following CTX injections. DAPI stained all nuclei (blue). An unpaired t-test showed **, p <0.01; ***, p<0.001. Error bars show the standard error of the mean (SEM).

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Immunostaining, Injection, Staining

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Gene ontology (GO) analysis reveals that numerous significantly downregulated genes in RNA samples from Jag2 sm vs. WT mouse MuSCs are muscle related. (B) Heatmap for down-regulated genes associated with myogenic regulatory genes, Notch receptor genes, and ligand genes. (C) GO analysis reveals that numerous significantly up-regulated genes in RNA samples from Jag2 sm vs. WT mouse MuSCs are involved in negative regulation of cell proliferation. (D) RT-qPCR was performed on WT and Jag2 sm MuSCs under growth, day 1, and day 3 differentiation conditions to detect the expression of myogenic and Notch pathway-related genes.

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Hes1-467 -Luc vector contains Hes1 467bp upstream promoter-driving luciferase ( Luc ) gene with an RBPJ binding site and Hes1-467-Mut-Luc vector contains a mutated RBPJ binding site. (B) Homozygous Jag2 sm MuSCs show higher Hes1-467-Luc activity compared with WT MuSCs. Luc activities were diminished when the RBPJ-binding site was mutated or when treated with the Notch inhibitor DAPT. (C) Expression of Notch1-4 ( N1-4 ) increased Hes1-467-Luc activities that were suppressed by co-transfection of human JAG2 in WT and homozygous Jag2 sm MuSCs. (D) Human JAG2- mediated suppression was not observed when transfected with expression vectors carrying pathogenic JAG2 variants. (E) 4R-SV-Luc contains 4 x E-boxes for consensus binding sites for MyoD. (F) Expression of MyoD and human JAG2 activates 4R-SV-Luc in WT and homozygous Jag2 sm MuSCs . MyoD promoted myosin heavy chain (MyHC)(+) myogenic differentiation in WT and homozygous Jag2 sm MuSCs in growth (G) and differentiation conditions (days 1 and 3). (H) Anti-Hes1 antibody staining shows Hes1 is higher in homozygous Jag2 sm compared with WT MuSCs. Hes1 expression is down-regulated in MyHC(+) myocytes (arrows). Scale bars, 50μm. (I) The diagram shows a DuoLink PLA assay to examine a protein complex of Jag2 and Notch1 within MuSCs. Anti-Jag2 and anti-Notch1 antibodies were used followed by anti-rabbit and anti-mouse IgG with (+) and (–) strands of oligo DNAs. Red fluorescence tags were incorporated with successful ligation. (J) DuoLink labeling shows patchy red complexes around the cell membrane regions, with no positive complexes when antibodies were eliminated as a control. (K) Quantification of DuoLink(+) complexes/cell was performed. DAPI stained all nuclei (blue). Scale bars, 100μm. An unpaired t-test showed *, p <0.05; **, p<0.01; ***, p<0.001. Error bars show the standard error of the mean (SEM).

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Plasmid Preparation, Luciferase, Binding Assay, Activity Assay, Expressing, Cotransfection, Transfection, Staining, Fluorescence, Ligation, Labeling, Membrane, Control

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: MuSCs isolated from homozygous Jag2 mice were used for expression vector-mediated human JAG2 overexpression. (A) Overexpression of WT human JAG2 ( JAG2 WT ) but not human JAG2 pathogenic variants ( p.Glu164Lys , p.Pro682Ser , and p.Phe977Ser ) increased MyHC(+) myogenic differentiation (green) (B), and fusion in days 1 and 3 differentiation conditions (C), compared with control empty vector-transfected cells. DAPI stained all nuclei (blue). Scale bars, 100 μm. An unpaired t-test showed *, p <0.05; **, p<0.01. Error bars show the standard error of the mean (SEM).

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Isolation, Expressing, Plasmid Preparation, Over Expression, Control, Transfection, Staining

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) MuSCs were layered on top of the MuECs with scrambled or Jag2 siRNA, allowed to adhere, and then co-cultured in differentiation medium for 5 days. (B) MuECs transfected with Jag2 siRNA show a significant reduction of Jag2 expression vs. scrambled siRNA. (C, D) Pax7(+)MyoD(-) self-renewing MuSCs were reduced when Jag2 was co-cultured with Jag2-KD MuECs vs. control MuECs (arrows). Downregulation of Notch signaling through the pan-Notch inhibitor DAPT reduced the number of Pax7(+)MyoD(-) self-renewing MuSCs vs. PBS-treated cells in the co-cultures (arrows). (E) Diagram of the evaluation of MuSCs treated with Notch ligands. (F) Hes1-467-Luciferase activity was assessed in control and JAG2 -expressing MuSCs exposed to Notch ligand (Control-IgG-Fc, Dll1-Fc, Dll4-Fc, Jag1-Fc, and Jag2-Fc). Comparative mRNA expression levels of the Notch effector genes (G) Hes1 , (H) Hey1 , and (I) HeyL , in control and JAG2 -expressing MuSCs exposed to Notch ligand (Control-IgG-Fc, Dll1-Fc, Dll4-Fc, Jag1-Fc and Jag2-Fc). DAPI stained all nuclei (blue). An unpaired t-test showed **, p<0.01; and ***, p<0.001. Error bars show the standard error of the mean (SEM). Scale bars, 50 μm.

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Cell Culture, Transfection, Expressing, Control, Luciferase, Activity Assay, Staining

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Following tamoxifen (TMX) injection, single or repeated CTX injections into the TA were performed. (B) H&E staining of TA by 7 days following CTX injection or 28 days following sequential CTX injections in Jag2 LoxP/LoxP :VEcad CreERT2 and Jag2 LoxP/LoxP :Pax7 CreERT2 mice. Scale bars, 100 μm. (C, D) Feret’s diameters of TA muscle fibers in Jag2 LoxP/LoxP :VEcad CreERT2 and Jag2 LoxP/LoxP :Pax7 CreERT2 mice following CTX injections. (E, F) Single muscle fibers were isolated at 28 days following TMX treatment and CTX injection. Anti-Pax7 antibody staining shows a reduced number of self-renewing MuSCs in Jag2 Δ/Δ :VEcad CreERT2 but not in Jag2 Δ/Δ :Pax7 CreERT2 mice. (G) MuSCs isolated from homozygous Jag2 Δ/Δ :Pax7 CreERT2 mice with TMX treatment show (G, H) reduced EdU(+) proliferating cells (green) in growth, (G, I) MyHC(+) myogenic differentiation (green), and (G, J) fusion index in day 1 and 3 of differentiation conditions compared with control cells. DAPI stained all nuclei (blue). Scale bars, 50 μm for (E) and 100 μm for (G). An unpaired t-test showed *, p<0.05; **, p<0.01; and ***, p<0.001. Error bars show the standard error of the mean (SEM).

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Injection, Staining, Isolation, Control

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: (A) Human JAG2 Ref induced delta-shaped wing veins,but variants showed marginal effects. (B) Serrate deficiency generated progressive melanotic spots on the legs. Expression of JAG2 Ref rescued manifestations of serrate deficiency, whereas expression of JAG2 E164K did not, on measures of (C) flight and (D, E) negative geotaxis, along with (F) melanotic spots. n=5 replicates, ***, p<0.001 (two-sided t-test).

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Generated, Expressing

Journal: bioRxiv

Article Title: JAG2 -related muscular dystrophy and Notch signaling dysfunction in muscle stem cells

doi: 10.1101/2025.07.23.665646

Figure Lengend Snippet: In mammals, neighboring capillary MuECs trans -activate Notch signaling in MuSCs via Jag2 for MuSC self-renewal. MuSCs, which do not receive Jag2 -mediated trans -activation by MuECs suppress Notch signaling via cis- inhibition by cell-autonomous Jag2 expression, stimulating myogenic differentiation. In Drosophila wing discs, the ortholog Serrate is expressed in epithelial cells, which activates Notch signaling in adjacent adult muscle precursors (AMPs), which are MuSC-like cells, to maintain the progenitor pool. AMPs express Serrate , but it is unclear whether cis- inhibition by Serrate occurs in AMPs.

Article Snippet: The Taqman probes used were: mouse Jag2 (Mm01325629_m1); mouse MyoG (Mm00446194_m1); human JAG2 (Hs99999198_m1), and mouse Gapdh (Mm99999915_g1) from ThermoFisher Scientific.

Techniques: Activation Assay, Inhibition, Expressing

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Schematic diagram of SKOV3-ip cell injection in mouse peritoneum. B Locations of (i) ascites, (ii) omentum, (iii) primary ovary, and (iv) diaphragm tumor progression 3- weeks after i.p. injection (left panel). H & E staining of omentum without (top right panel) and with tumor (right bottom panel). C Endpoint analysis of malignant ascites and total number of tumor nodules in omentectomized mice 4 weeks after intraperitoneal cell injection of SKOV3-ip cells. Data are shown as Mean +/-SEM ( n = 3). Statistical significance was calculated using Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparison test. ** P < 0.01, **** P < 0.0001. D–F Jagged2 protein and RNA expression in whole mouse omentum three weeks following in vivo i.p. injection of SKOV3-ip tumor cells. Immunofluorescence ( D ), Immunoblot ( E ), and qRT-PCR (F) analysis were performed using Jagged2 specific antibody and probe (** P < 0.01; Scale bars 100 μm). G Diagram of the mesothelium in 3D culture. Primary human omental fibroblasts are plated with human omental ECM and cultured for 6 hours. The fibroblasts are overlaid with human omental mesothelial cells and cultured for an additional 24 hours before OvCa cells are seeded. The purpose of the use of the 3D culture system was to investigate OvCa cells’ adhesion, invasion, and proliferation. H Ex-vivo human omentum adhesion, invasion, and proliferation assay with PHK25 labeled OvCa cells and the schematic of cell sorting by FACS. I Western blot analysis of Jagged2 protein in the surface cells of the human omentum in two culture conditions. Cells were cultured in human omentum without fluorescently labeled cells (unattached) or PHK25 labeled SKOV3-ip cells (attached) after FACS sorting. J qRT-PCR mRNA analysis of Jagged2 following the same methods described in H (** P < 0.01).

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Injection, Staining, Comparison, RNA Expression, In Vivo, Immunofluorescence, Western Blot, Quantitative RT-PCR, Cell Culture, Ex Vivo, Proliferation Assay, Labeling, FACS

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Heat map showing the qRT-PCR gene expression of the Notch pathway ligands and downstream targets in four ovarian cancer cell lines. B qRT-PCR analysis of the Jagged2 gene expression in ovarian cancer cell lines with distinct metastatic abilities. C Heat map showing the microarray data analysis of the Jagged2 mRNA expression in ovarian cancer cell lines with metastatic ability. D – F Kaplan-Meier progression-free survival curves of ovarian cancer patients from Gentric (GSE26193) , Denkert (GSE14764) , and TCGA data sets representing high and low expression of Jagged2. G Western blot analysis of the Jagged2 protein in control and Jagged2 knockdown (KD) in OVCAR3 and SKOV3-ip cells. H (Top) Representative images of tumor nodules in mouse metastatic omentum (right) and tumors developed in the primary ovary (left). (Bottom) quantification of tumor area from each group. Scale bar 100 μm. *** P < 0.001. I–L Kaplan-Meier survival and omental metastasis- free curves of mice injected with control or Jagged2 knockdown (Jag2KD) OVCAR3 and SKOV3-ip cells (n = 5 mice per/group). M (Left panel) Representative immunohistochemical images of Ki-67 and H & E histological images of primary omentum from representative mice for each experimental group. (Right panel) quantification of Ki-67 -positive cells in the Jag2KD cells compared with the control group. Scale bar 20 μm. P = 0.12 (OVACR3 cells) and 0.44 (SKOV3-ip cells). Data in the figure represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Quantitative RT-PCR, Expressing, Microarray, Western Blot, Control, Knockdown, Injection, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Western blot analysis showing Jagged2 protein expression in control and Jag2OE OV2774 cell line. B Mice inoculated with control and Jag2OE cells showed the absence ( n = 5) and presence ( n = 5) of omental tumor nodules with histological and Jagged2 immunofluorescence. Magnification of images are taken at 40x (scale bar = 200 μm). C Quantification of tumor area in the omentum from each mice group ( P = 0 .0019). Error bar represent Mean +/- SD; *** P < 0.001. D Quantification of tumor weight (in grams) from each mice group from the day of inoculation (0 days) up to day 25. Error bar represent Mean +/- SD; ns - not significant; * P < 0.05; ** P < 0.01 and *** P < 0.001, respectively. E Kaplan-Meier omental metastasis-free survival curve of mice inoculated with control ( n = 5) or Jag2OE ( n = 5) (Log-rank P < 0.012). F Representative images of freshly resected human omentum showing absence (no omental metastasis; n = 4) and presence (omental metastasis; n = 4) of omental tumors with histological analysis of both H & E and Jagged2 immunofluorescence. Scale bar 100 μm. G Immunofluorescence staining of Ki-67 from omental metastatic tumors from each group (left panel). The right panel illustrates the quantification of the percent of Ki-67 -positive tumor cells. For the quantification analysis, 10 distinct regions were randomly selected from each group. * P < 0.05. H (Left panel) Representative images of invaded cells and (right panel) quantification of cells invasive capability of control and Jag2OE cells using two-chamber transwell Matrigel invasion assay. ** P < 0.01. Scale bar 100 μm, I qRT-PCR mRNA expression analysis of the Notch target genes Hes1 (** P < 0.01), Hey1 (*** P < 0.001), and the Notch receptor Jagged2 (** P < 0.01) in control and Jag2OE OV2774 mice omental metastatic site. Data in the figure represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Invasion Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Gene-set enrichment analysis (GSEA) of the TGF-β-responsive genes set in a ranked list of differentially expressed genes in primary ovary tumors versus omental metastatic tumor cells (GSE2109; P = 0.037). The bottom panel shows a corresponding heat map of core TGF-β gene sets with elevated expression in two groups. B Jagged2 mRNA expression in response to TGF-β treatment in ovarian cancer cell lines. ** P < 0.01, *** P < 0.001. C Jagged2 mRNA expression in SKOV3-ip cells treated with either DMSO or TGF-β-receptor inhibitor (EMD616451) for 24 hours. *** P < 0.001. D Cell proliferation assay of SKOV3-ip cells treated with either DMSO or TGF-β-receptor inhibitor (EMD616451) for the indicated time. * P < 0.05, *** P < 0.001. E Jagged2 mRNA expression in ovarian cancer cells treated with either DMSO or Smad3 inhibitor (SIS3) for 24 hours. *** P < 0.001. F Number of invaded cells in the indicated cells with altered expression of Jagged2 and Smad3. ** P < 0.01, *** P < 0.001. G Representative images of the omental region in mice from experimental groups on day 10. (left) Quantification of tumor numbers in each group of experimental mice. Data in the figure represent average +/- SEM, SD. ** P < 0.01, *** P < 0.001. H Western blot analysis showing Jagged2, Smad3, and phospho-Smad3 expression in the indicated cells with altered Jagged2 and Smad3. I Western blot analysis of Jagged2, phospho-Smad3, and Smad3 protein levels in the control or Smad3 knockdown in Jag2OE cells with or without stimulation of TGF-β. J Western blot analysis showing Jagged2 protein expression in the control and Jag2KD SKOV3-ip cells with or without TGF-β treatment. Data in the figure represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Expressing, Proliferation Assay, Western Blot, Control, Knockdown

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Schematic diagram of a co-culture model between Jag2OE tumor cells and human primary mesothelial cells transfected with a Notch reporter. B Flow cytometry cells separation of mesothelial cells (P2) from GFP+ Jag2OE cells (P3). After flow cytometry cell separation, the bottom panel shows the quantification of the Jagged2 mRNA expression control and sorted co-culture cells from the indicated group (*** P < 0.001). C Co-culture between control or Jag2OE tumor cells and mesothelial cells transfected with a Notch reporter and treated with DMSO or MRK-003 (1 and 5 μM). D qRT-PCR assessment of the mRNA levels of the indicated Notch target genes, TGF-β1 and fibronectin 1 (FN1) in mesothelial cells separated by FACS from co-culture in each experimental group. * P < 0.05, ** P < 0.01, and *** P < 0.001. E qRT-PCR analysis of Jagged2 mRNA expression in primary human mesothelial cells with or without a conditioned medium (CM) from Jog2OE cells for the indicated time periods. * P < 0.05. F Representative images of co-cultures of Jag2OE and mesothelial cells from each experimental group (DMSO or MRK-003 [5 μM]). Scale bar, 200 μm. G Quantifying tumor cell proliferation of control and Jag2OE cells after co-culture with mesothelial cells from each experimental group by luciferase assay. ** P < 0.01, *** P < 0.001. H Quantification of sphere growth efficiency of control and Jag2OE after co-cultures of each experimental treatment group. ** P < 0.01, *** P < 0.001. I Quantification of tumor cell proliferation cultured alone (no co-culture) from each experimental group. Data in the figure represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated. ns = not significant. All experiments were run in triplicates.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Co-Culture Assay, Transfection, Flow Cytometry, Expressing, Control, Quantitative RT-PCR, Luciferase, Cell Culture

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Jagged2 expression was examined by fluorescent-activated cell sorting (FACS) in tissues from omentum ( n = 4) sampled from patients treated for benign disease, OvCa primary tumors ( n = 4), and omental metastasis ( n = 4) collected from patients with serous OvCa. Metastatic tumors in the omentum ( n = 4) had a higher percentage of Jagged2+ cells compared with patients who were either primary tumors or benign omentum ( n = 4). ** P < 0.01, *** P < 0.001 unpaired two-sided t -test. B Spheroids formation assay comparing cultured omental metastatic (Jag2High) and non-metastatic omentun (Jag2Low) cells. Cells were seeded in ultra-low adherence 96-well plates, and the formation of spheroids was assessed with a wide-filed microscope. (Mean SEM + /-). Scale bar 200 μm. Jagged2 protein immunofluorescently stained with Jagged2 specific primary antibody in cultured spheroids. (Mean SEM + /-). *** P < 0.001, unpaired two-sided t -test. C (Left)Representative images of tumor localization in mouse omentaum at day 10 after injection of control and Jag2OE cells. (Right) Analysis of tumor burden in omentum at day 10 after injection. Data are presented as mean +/-SD ( n = 3) and a statistically significant difference was calculated using the Man-Whitney U test, *** P < 0.001. D Sphere-formation assay of SKOV3-ip and Jag2OE cells after mesothelial cell exposure. Representative sphere images and quantification of sphere numbers fold increase is shown ( n = 3). Scale bar 200 μm. A statistically significant difference was calculated using the Man-Whitney U test, *** P < 0.001. E In vivo limiting dilution assay showing tumor formation rate of Jag2OE cells co-injected with mesothelial cells ( n = 3 mouse/group). F Number of spheres and sphere diameter ( n = 3) in the indicated treatment group. ** P < 0.01, *** P < 0.001. G qRT-PCR analysis showing relative mRNA expression of the stemness markers, Sox2, Nanog, Oct3/4 in the indicated treatment group. One-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001. H ALDH1L1 mRNA in Jag2OE cells after coculture with mesothelial cells and in the indicated treatment groups as normalized to GAPDH mRNA ( n = 3). * P < 0.05, ** P < 0.01. I (right) Schematic presentation of the drug response assay designed to assess the proportion of apoptotic cells among the co-cultured OvCa and mesothelial cells. (left) GFP-labeled Jag2OE cells isolated from mesothelial cells were stained with Annexin-V and 7-AAD and the proportion of Annexin-V positive cells was determined by flow cytometry. J The percentage of Annexin-V-7AAD positive cells were presented from paired samples ( n = 8, P = 0.0078). p -values were analyzed based on Wilcoxon test. Data in the figure represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Expressing, FACS, Tube Formation Assay, Cell Culture, Microscopy, Staining, Injection, Control, In Vivo, Limiting Dilution Assay, Quantitative RT-PCR, Labeling, Isolation, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A A List of genes with an expression fold change between pleural malignant and peritoneal malignant mesothelial cells of more than 2-fold from the public dataset microarray GSE63966. B qRT-PCR mRNA analysis of the Notch target genes, and CTGF and FN1 from mesothelial cells, omental metastatic tumor cells, and primary ovary tumor cells resected from the patient. C (Left) Schematic model of the co-culture system of mesothelial and Jag2OE cells. (Right) qRT-PCR mRNA expression of the indicated genes in control and Jag2OE tumor cells cocultured with mesothelial cells. * P < 0.05, ** P < 0.01, *** P < 0.001. D Heat map showing qRT-PCR mRNA expression levels of the indicated genes from mesothelial cells that were FACS separated from cocultures of each experimental/treatment group. E qRT-PCR analysis of Hes1 expression in mesothelial cells treated with scrambled or Hes1 siRNA and cultured in 24 well plates coated with either Fc or control or recombinant Jagged2 protein ( n = 3). Data represent average +/- SD. ** P < 0.01, *** P < 0.001. F Quantification of cell proliferation in mesothelial cells treated with control or Hes1 siRNA and cultured in 24 well plates coated with either Fc or control or recombinant Jagged2 protein by luciferase assay ( n = 3). ** P < 0.01, *** P < 0.001. G Quantification of IL-6 levels in the conditioned media of control or Jag2OE tumor cells cultured alone or cocultured with mesothelial cells in the presence of DMSO, MRK-003 (1μM) by ELISA ( n = 3). ** P < 0.01, **** P < 0.001. H Quantification of IL-6 levels in the conditioned media of the indicated tumor cells co-cultured with mesothelial cells after treatment with Hes1 siRNA by ELISA. *** P < 0.001. I Cell proliferation of the indicated tumor cells co-cultured with mesothelial cells from each experimental group by luciferase assay. Data represent average +/-SD, ** P < 0.01, *** P < 0.001. J Cell proliferation of the indicated tumor cells cocultured with mesothelial cells after treatment with Hes1 siRNA by luciferase assay. ** P < 0.01, *** P < 0.001. K Quantification of sphere formation capacity of the indicated tumor cells cocultured with mesothelial cells after treatment with Hes1 siRNA. ** P < 0.01, *** P < 0.001. L Quantification of sphere formation capacity of indicated tumor cells cocultured with mesothelial cells after treatment with 1 μM MRK-003. ** P < 0.01, *** P < 0.001. M Cell proliferation of indicated tumor cells cocultured with mesothelial cells and treatment with either IgG or 5.0 μg/mL of the anti-IL-6 antibody by luciferase assay. ** P < 0.01, *** P < 0.001. N Cell proliferation of indicated tumor cells cocultured with mesothelial cells and treatment with either PBS, 5.0 μg/mL, or 10.0 μg/mL of anti-IL6 by luciferase assay. * P < 0.05, *** P < 0.001. Data in the figure represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated. All experiments were run in triplicates.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Co-Culture Assay, Control, Cell Culture, Recombinant, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A , B OvCa cells were treated with vehicle and MRK-003 (5 μM), followed by the addition of fluorescently labeled SKOV3-ip and OVCAR3 cells and later detected by fluorescent reader. ** P < 0.01, *** P < 0.001. C The experimental model of vehicle or MRK-003 (5 μM) treated and fluorescently labeled OvCa cells seeded in a piece of human omentum (72 hours). D–E Jagged2 inhibition by MRK-003 as described in ( A and B ). After scraping off the omentum surface cells of the omentum, Jagged2 ( D ) and CD44 ( E ) were quantified using qRT-PCR. * P < 0.05, *** P < 0.001. F Quantification of the number of cells after scrapping from the omentum. * P < 0.05, ** P < 0.01. G - H qRT-PCR mRNA analysis of the Notch target genes and IL-6 from scraped-off cells from the omentum. * P < 0.05, ** P < 0.01, *** P < 0.001. I Kaplan-Meier omental metastasis-free survival curve of mice from each experimental group. (Log-rank P = 0.018). J Representative images of tumor metastasis to omentum (top; highlighted with white circles) and primary ovary site (bottom) in female mice injected with SKOV3-ip cells and treated with vehicle or MRK-003. K–N Omental tumor weight ( K ), omental tumor number ( L ), ascites volume ( M ), and omental weight ( N ) at the endpoint of mice intraperitoneal inoculation. P = 0.006 and 0.009; * P < 0.05, *** P < 0.001. O qRT-PCR analysis of the Notch target genes and IL-6 in the tumor-stromal compartment of omental metastasis from vehicle or MRK-003 treated mice. * P < 0.05, ** P < 0.01, *** P < 0.001. Data in the figure represent average and +/-SEM; P values were determined using the Student’s t -test unless otherwise indicated. All experiments were run in triplicates.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Labeling, Inhibition, Quantitative RT-PCR, Injection

Journal: Cell Death & Disease

Article Title: Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells

doi: 10.1038/s41419-024-06512-0

Figure Lengend Snippet: A Schematic diagram of i.p. cell injection and treatment schedules. B Images of representative mice in each experimental group on day 40 after peritoneal injection. C Kaplan-Meier omental metastasis-free survival curve of mice from each experimental group. Log-rank P = 0.0032. D–F Omental tumor weight ( D ), omental tumor number ( E ), and ascites volume ( F ) of mice from each experimental group (mean +/- SEM, n = 5 for each experiment, ** P < 0.01, *** P < 0.001, Students t - test). G A graphical presentation of tumor-mesothelial cell cross-talk of Jagged2-expressing ovarian tumor cells with the omental microenvironment. Data in the figures represent average and +/-SEM; P -values were determined using the Student’s t -test unless otherwise indicated. All experiments were run in triplicates.

Article Snippet: JAG2 (cat#TL303860) human 4-unique shRNA constructs in lentiviral GFP vector were purchased from Origene (OriGene Technologies, USA).

Techniques: Injection, Expressing