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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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:

Article Snippet: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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) 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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

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: Jag2 siRNAs (sc-39673, Santa Cruz Biotechnology) and control scramble siRNA-A (sc-37007, Santa Cruz Biotechnology) were transfected in ECs by Polyjet (11668019, ThermoFisher Scientific) before co-culture.

Techniques: Activation Assay, Inhibition, Expressing

Journal: Cell Death & Disease

Article Title: CD146 regulates the stemness and chemoresistance of hepatocellular carcinoma via JAG2-NOTCH signaling

doi: 10.1038/s41419-025-07470-x

Figure Lengend Snippet: Primer sequences information in the study.

Article Snippet: Antibodies for western blotting included mouse anti-β-Actin (1:1000), mouse anti-GADPH (Proteintech, Cat#60004-1-IG, 1:1000), rabbit anti-CD146 antibody (Abcam, Cat#ab75769, 1:1,000), rabbit anti-Jagged2 (JAG2) (Cell Signaling Technology, Cat#2210, 1:1,000), rabbit anti-Notch1 (NOTCH1) (Proteintech, Cat#20687-1-AP, 1:1,000), and rabbit anti-HES1 (ABclonal, Cat#A0925, 1:1,000).

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: CD146 regulates the stemness and chemoresistance of hepatocellular carcinoma via JAG2-NOTCH signaling

doi: 10.1038/s41419-025-07470-x

Figure Lengend Snippet: A , B RT-qPCR analysis of Notch signaling pathway genes in CSQT-2-Ctrl and CSQT-2-CD146 cells ( A ) or shCtrl and shCD146 cells ( B ). The protein levels of JAG2, NOTCH1 and HES1 in CSQT-2-Ctrl and CSQT-2-CD146 cells ( C ) or shCtrl and shCD146 cells ( D ). E , F RT-qPCR analysis of JAG2, NOTCH1 and HES1 in shCtrl, shCD146 cells, shCtrl with stably JAG2 overexpression, shCD146 cells with stably JAG2 overexpression. G NOTCH1 and HES1 in shCtrl, shCD146 cells, shCtrl with stably JAG2 overexpression, shCD146 cells with stably JAG2 overexpression were detected by western blotting. H JAG2, NOTCH1 and HES1 in CSQT-2-Ctrl and CSQT-2-CD146 cells treated with DMSO or QNZ(EVP4593) (NF-κB signaling inhibitor, 5 μM) for 24 h were detected by western blotting. I Western blot analysis of p65 in the cytoplasm and nucleus of CSQT-2-Ctrl and CSQT-2-CD146 cells. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant).

Article Snippet: Antibodies for western blotting included mouse anti-β-Actin (1:1000), mouse anti-GADPH (Proteintech, Cat#60004-1-IG, 1:1000), rabbit anti-CD146 antibody (Abcam, Cat#ab75769, 1:1,000), rabbit anti-Jagged2 (JAG2) (Cell Signaling Technology, Cat#2210, 1:1,000), rabbit anti-Notch1 (NOTCH1) (Proteintech, Cat#20687-1-AP, 1:1,000), and rabbit anti-HES1 (ABclonal, Cat#A0925, 1:1,000).

Techniques: Quantitative RT-PCR, Stable Transfection, Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: CD146 regulates the stemness and chemoresistance of hepatocellular carcinoma via JAG2-NOTCH signaling

doi: 10.1038/s41419-025-07470-x

Figure Lengend Snippet: A RT-qPCR analysis of Oct-4, EpCAM, Nanog were carried out in CSQT-2-Ctrl and CSQT-2-CD146 cells treated with DMSO or RO4929097 (Notch signaling pathway inhibitor, 10 μM) for 24 h. B Sphere formation ability of CSQT-2-Ctrl and CSQT-2-CD146 cells treated with DMSO or RO4929097 (10 μM) for 24 h. Scale bars = 120 μm. C CSQT-2-Ctrl and CSQT-2-CD146 cells were treated with DMSO or RO4929097 (10 μM) for 24 h, following were treated with indicated concentrations of cisplatin for 48 h. CCK-8 assay was used to measure cell viability. D RT-qPCR analysis of Oct-4, EpCAM were executed in shCtrl, shCD146, shCtrl with stably JAG2 overexpression, shCD146 cells with stably JAG2 overexpression. E Comparison of sphere formation ability between shCtrl, shCD146, shCtrl with stably JAG2-overexpressing, shCD146 cells with stably JAG2 overexpression by accessing number of tumor spheres (7 days). Scale bars = 120 μm. F The cell lines of shCtrl, shCD146#1, shCD146#2, shCD146#2 with control virus, shCD146#2 with stably CD146 overexpression, shCD146#2 with stably JAG2 overexpression PLC/PRF/5 cells were exposed to cisplatin at the concentration corresponding to its IC50 value for 48 h. The cell lines of shCtrl, shCD146, shCtrl with stably JAG2 overexpression, shCD146 with stably JAG2 overexpression Huh7 cells were treated with indicated concentrations of cisplatin for 48 h. CCK-8 assay was used to measure cell viability. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001; ns not significant).

Article Snippet: Antibodies for western blotting included mouse anti-β-Actin (1:1000), mouse anti-GADPH (Proteintech, Cat#60004-1-IG, 1:1000), rabbit anti-CD146 antibody (Abcam, Cat#ab75769, 1:1,000), rabbit anti-Jagged2 (JAG2) (Cell Signaling Technology, Cat#2210, 1:1,000), rabbit anti-Notch1 (NOTCH1) (Proteintech, Cat#20687-1-AP, 1:1,000), and rabbit anti-HES1 (ABclonal, Cat#A0925, 1:1,000).

Techniques: Quantitative RT-PCR, CCK-8 Assay, Stable Transfection, Over Expression, Comparison, Control, Virus, Concentration Assay

Journal: Cell Death & Disease

Article Title: CD146 regulates the stemness and chemoresistance of hepatocellular carcinoma via JAG2-NOTCH signaling

doi: 10.1038/s41419-025-07470-x

Figure Lengend Snippet: A Correlation analysis of CD146 and the key genes of Notch signaling pathway (JAG2 and NOTCH1) in the TCGA database (HCC). B Left: IHC analysis of CD146 and JAG2 expressions in human HCC samples. Scale bars = 100 μm. Right: Correlation analysis of CD146 and JAG2 in human HCC samples. C Kaplan-Meier survival curve of overall survival (OS) for patients with CD146 high JAG2 high and CD146 low JAG2 low in HCC patients from the GSE database (GSE14520). D The signaling pathway pattern diagram for CD146 regulates HCC stemness.

Article Snippet: Antibodies for western blotting included mouse anti-β-Actin (1:1000), mouse anti-GADPH (Proteintech, Cat#60004-1-IG, 1:1000), rabbit anti-CD146 antibody (Abcam, Cat#ab75769, 1:1,000), rabbit anti-Jagged2 (JAG2) (Cell Signaling Technology, Cat#2210, 1:1,000), rabbit anti-Notch1 (NOTCH1) (Proteintech, Cat#20687-1-AP, 1:1,000), and rabbit anti-HES1 (ABclonal, Cat#A0925, 1:1,000).

Techniques:

Journal: eLife

Article Title: Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway

doi: 10.7554/eLife.95267

Figure Lengend Snippet:

Article Snippet: Antibody , mouse monoclonal anti-Jagged2 , Santa Cruz , #sc-515725 , WB (1:100).

Techniques: