jag2  (Sino Biological)

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Machine learning identification of key genes. (A) Venn diagram of differentially expressed genes (DEGs) and weighted gene co‐expression network (WGCNA) module genes; (B) least absolute shrinkage and selection operator regression (LASSO) coefficient selection plot; (C) random forest (RF) identification of key factors, with mean decrease accuracy (left) indicating the importance of genes to model prediction accuracy and mean decrease Gini (right) indicating the importance of genes to model classification purity; (D) Venn diagram of LASSO and RF analysis; (E) box plot of Jag2 and Pcdh19 expression in transcriptome datasets; (F) receiver operating characteristic (ROC) curve of Jag2 in transcriptome datasets, with Y ‐axis representing sensitivity and X ‐axis representing specificity. Normoxia: n = 7; Hypoxia: n = 7.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Expressing, Selection

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Effects of NADPH oxidase 2 (NOX2) overexpression on proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. (A) Expression of Jag2 and NOX2 in PASMCs at various time points under hypoxic treatment detected by immunofluorescence. (B) mRNA levels of Jag2 and NOX2 in PASMCs at various time points under hypoxic treatment detected by qRT‐PCR. (C) Cell proliferation levels were measured by the CCK‐8 assay. (D) Cell proliferation levels assessed by EdU staining, scale bar = 50 μm. (E) Protein expression of Jag2, NOX2, proliferating cell nuclear antigen (PCNA), and survivin in different groups was detected by the western blot. (F) Cell migration levels were assessed using a Transwell assay. (G) Protein expression of nuclear factor erythroid 2–related factor 2 (Nrf2) and SOD2 in different groups detected by the western blot; levels of superoxide dismutase (SOD), malondialdehyde (MDA), total glutathione (GSH), reduced GSH, and oxidized GSH measured by assay kits; reactive oxygen species (ROS) levels assessed by 2',7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining. (H) Protein expression of caspase‐3, cleaved caspase‐3, B‐cell lymphoma 2 (Bcl2), and Bcl‐2‐associated X protein (BAX) in different groups detected by the western blot. (I) Apoptosis levels in cells of different groups were assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, scale bar = 50 μm. (J) Apoptosis levels in cells of different groups were measured by flow cytometry (FCM). * indicates p < 0.05 compared to the hypoxia + siJag2 group, ** indicates p < 0.01 compared to the hypoxia + siJag2 group; all cell experiments were performed in triplicate.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Over Expression, Migration, Expressing, Immunofluorescence, Quantitative RT-PCR, CCK-8 Assay, Staining, Western Blot, Transwell Assay, Measured Assay, TUNEL Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Mechanistic study of Jag2 promoting pulmonary artery smooth muscle cell (PASMC) proliferation and migration under hypoxic conditions through the NADPH oxidase 2 (NOX2)/reactive oxygen species (ROS) pathway. (A) CCK‐8 assay to measure cell proliferation levels in each group; (B, C) EdU staining to measure cell proliferation levels in each group (B), with panel C showing the bar graph statistical results of panel B, scale bar = 50 μm; (D) western blot analysis of Jag2, NOX2, proliferating cell nuclear antigen (PCNA), and survivin protein expression in each group; (E) Transwell assay to measure migration levels in each group; (F) western blot analysis of nuclear factor erythroid 2–related factor 2 (Nrf2) and SOD2 protein expression in each group; (G) kit assays to measure superoxide dismutase (SOD) and malondialdehyde (MDA) levels in each group; (H) kit assays to measure total glutathione (GSH), reduced GSH, and oxidized GSH levels in each group; (I) 2',7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining to measure ROS levels in each group; (J) western blot analysis of caspase‐3, cleaved caspase‐3, B‐cell lymphoma 2 (Bcl2), and Bcl‐2‐associated X protein (BAX) expression in each group; (K) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to measure apoptosis levels in each group, scale bar = 50 μm; (L) flow cytometry (FCM) to measure apoptosis levels in each group. * p < 0.05 compared to the normoxia group, ** p < 0.01 compared to the normoxia group, # p < 0.05 compared to the hypoxia + siNC group, ## p < 0.01 compared to the hypoxia + siNC group. All experiments were repeated three times.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Migration, CCK-8 Assay, Staining, Western Blot, Expressing, Transwell Assay, TUNEL Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Regulation of gene expression and vascular remodeling by the Jag2/NADPH oxidase 2 (NOX2) pathway in hypoxic pulmonary arterial hypertension (PAH) rat models. (A) Measurement of mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), pulmonary artery systolic pressure (PASP), and right ventricle (RV)/(left ventricle [LV] + S) values in each group of rats. (B) H&E and Elastica Van Gieson (EVG) staining of pulmonary artery remodeling in each group of rats (scale bar = 50 μm). (C) Transmission electron microscopy of the basal membrane morphology of pulmonary arterioles in each group of rats (scale bar = 5 μm). (D) Western blot analysis of Jag2, NOX2, alpha‐smooth muscle actin (α‐SMA), vimentin, CD31, and VE‐cadherin expression in each group. (E) Immunofluorescence detection of CD31 and α‐SMA expression in the pulmonary arteries of each group of rats (scale bar = 50 μm). (F) Western blot analysis of proliferating cell nuclear antigen (PCNA) and survivin expression in lung tissues of each group. *indicates p < 0.05 compared to the control group; ** indicates p < 0.01 compared to the control group; # indicates p < 0.05 compared to the model + AAV‐shNC group; ## indicates p < 0.01 compared to the model + AAV‐shNC group. N = 8.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Gene Expression, Staining, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Expressing, Immunofluorescence, Control

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Effects of the Jag2/NADPH oxidase 2 (NOX2) pathway on inflammation, oxidative stress, and apoptosis in a hypoxic pulmonary arterial hypertension (PAH) rat model. (A) Immunohistochemistry detection of CD68 expression in the lungs of different groups of rats, scale bar = 50 μm; (B) ELISA detection of tumor necrosis factor‐alpha (TNF‐α) and IL‐6 levels in serum and lung tissue; (C) measurement of superoxide dismutase (SOD) and malondialdehyde (MDA) levels in the serum of different rat groups; (D) western blot detection of nuclear factor erythroid 2–related factor 2 (Nrf2) and SOD2 expression in lung tissue; (E) DHE staining for reactive oxygen species (ROS) levels in lung tissue, scale bar = 50 μm; (F) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection of apoptosis levels in lung tissue, scale bar = 20 μm. * p < 0.05 compared to the control group, ** p < 0.01 compared to the control group, # p < 0.05 compared to the model + AAV‐shNC group, ## p < 0.01 compared to the model + AAV‐shNC group, N = 8.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Immunohistochemistry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, TUNEL Assay, 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) 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:

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:

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: 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:

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:

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