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: Nature Immunology

Article Title: Exclusion of Notch from the contact site during efferocytosis restricts anticancer immunity

doi: 10.1038/s41590-026-02452-3

Figure Lengend Snippet: Kaplan-Meier survival curves obtained from all TCGA solid tumor studies (n = 10720). ( a and b ) Survival outcomes in patients with low (bottom 25%) or high (top 25%) expression of Rubicon and high ( a ) or low ( b ) expression of DLL1 . (cand d) Survival outcomes in patients with low (bottom 25%) or high (top 25%) expression of Rubicon and high ( c ) or low ( d ) expression of DLL4 . ( e and f ) Survival outcomes in patients with low (bottom 25%) or high (top 25%) expression of Rubicon and high ( e ) or low ( f ) expression of JAG1 . ( g and h ) Survival outcomes in patients with low (bottom 25%) or high (top 25%) expression of Rubicon and high ( g ) or low ( h ) expression of JAG2 . Statistical significance evaluated using the Log-rank test.

Article Snippet: We tested whether efferocytosis by Rubcn-deficient BMDMs induced proteolytic activation of the Notch receptor by coculturing apoptotic HT115 cells that express Notch ligands JAG1 and JAG2 (The Human Protein Atlas) with BMDMs and performed nuclear fractionation to assess the translocation of the N2ICD by immunoblotting.

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Ultra-low-input rG4-seq reveals the RNA G-quadruplex regulome in gene expression and genome integrity

doi: 10.1093/nar/gkag040

Figure Lengend Snippet: DHX36 regulates transcriptional elongation through modulation of RNA G-quadruplex structures. ( A and B ) Representative images (A) and quantification (B) of nascent transcription visualized by EU incorporation (green) in control and Dhx36 fl/fl ;SKO oocytes. Scale bars: 10 μm. ( C and D ) Representative images (C) and quantification (D) of RNA Polymerase II (Pol II, green) in control and Dhx36 fl/fl ;SKO oocytes; scale bars: 10 μm. ( E and F ) Metagene analysis showing Pol II distribution around TSS and TES in control versus Dhx36 fl/fl ;SKO oocytes ( e ) and control versus cPDS-treated oocytes (F). ( G ) Heatmap showing genome-wide Pol II distribution patterns in control, Dhx36 fl/fl ;SKO , and cPDS-treated oocytes. ( H ) Cumulative distribution of Pol II pausing index in control and Dhx36 fl/fl ;SKO oocytes. Inset shows boxplot of log2 pausing index. ( I ) Bar graph showing the number of up- and down-regulated transcription elongation-related genes with rG4 structures. ( J and K ) Genome browser tracks showing rG4 signals and DHX36 binding at the Ccnt1 (J) and Tcea1 (K) loci. ( L and M ) Representative images (L) and quantification (M) of CCNT1 protein in NSN and SN stage oocytes from control and Dhx36 fl/fl ;SKO mice. ( N and O ) Representative images (N) and quantification (O) of TCEA1 protein in control and Dhx36 fl/fl ;SKO oocytes. ( P–R ) Representative images (P) and quantification (Q and R) of phosphorylated Ser2 RNA Pol II (pS2) and CDK9 in control and Dhx36 fl/fl ;SKO oocytes. ( S ) Metagene analysis showing CDK9 distribution around TSS and TES in control and Dhx36 fl/fl ;SKO oocytes. ( T ) Schematic model illustrating DHX36-mediated regulation of transcriptional elongation through rG4 unwinding. Data are presented as mean ± SEM; P < 0.0001 (Student’s t -test); scale bars: 10 μm.

Article Snippet: Primary antibodies used were: SRSF1/ASF/SF2 (12929-2-AP, Proteintech, rabbit, IF), RNASEH1 (15606-1-AP, Proteintech, rabbit, IF/ICC), Phospho-Histone H2A.X (Ser139) (20E3, 9718S, CST, rabbit, IF), 53BP1 (ET1704-05, HUABIO, rabbit, IF), YBX2/MSY2 (R1510-36, HUABIO, rabbit, IF), DHX36 (ab70269_50 μl, Abcam, WB), Anti-DNA G-quadruplex (G4) (clone 1H6, MABE1126, Merck Millipore, mouse, IF), Anti-GFP (ab290, Abcam, rabbit, LACE-seq), Anti-FLAG M2 (F1804-50UG, Sigma, LACE-seq), HA-tag (MF5) (H1003-100 μl, Lablead, mouse, LACE-seq), RanBP16/exportin 7 (GTX638871, Genetex, rabbit, IF), RNA pol II (39 097, Active Motif, mouse, IF/Cut&Tag), RNA pol II CTD phospho Ser2 (Clone 3E10, 61 984, Active Motif, rat, IF), Anti-DNA-RNA Hybrid (clone S9.6, MABE1095, Merck Millipore, mouse, IF), SFRS9 (A12538, Abclonal, rabbit, IF), HEXIM1 (ab240647, Abcam, rabbit, Cut&Tag), Cdk9 (ab239364, Abcam, rabbit, IF/Cut&Tag), p300 (F-4, sc-48343, Santa Cruz Biotechnology, mouse, IF), Cyclin T1 ( R24032 , Zen-bio, rabbit, IF), TCEA1 ( A20972 , Abclonal, rabbit, IF), and DDX4/MVH antibodies (ab13840 and ab27591, Abcam, rabbit and mouse respectively, IF).

Techniques: Control, Genome Wide, Binding Assay