one step tunel assay kit  (Proteintech)

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Cerebral I/R induces transient activation of ER stress transmembrane sensors, followed by apoptosis. (A) Schematic representation of the timeline of the experimental procedure. Mice were subjected to sham/MCAO for 1.5 hours followed by recovery/reperfusion. Brain tissues were collected at various time points during reperfusion for Western blotting, and brain infarction and neurological evaluation were performed after 24 hours recovery/reperfusion. (B) Triphenyl-tetrazolium chloride–stained cerebral coronal sections from representative brains, collected at 24 hours after reperfusion. The infarcted area is shown in white, and the normal area is shown in red. (C) Statistical analysis of the infarct volumes ( n = 5). **** P < 0.0001, I/R group vs . sham group, unpaired t- test. (D) Statistical analysis of the neurological scores 24 hours after reperfusion by Zea-Longa test ( n = 5). **** P < 0.0001, I/R group vs . sham group, Mann–Whitney U test. (E–J) The ischemic brain tissues were collected after 1.5 hours of MCAO (reperfusion 0 hours) or 1, 3, 6, 12, and 24 hours after reperfusion. The tissues from the sham group were collected at 1.5 hours after the sham surgery. (E) Representative western blot images of p-eIF2α, p-IRE1, ATF6, ATF4, and CHOP at different time points ( n = 3) after reperfusion. (F–J) Quantifications of the normalized levels of p-eIF2α/eIF2α (F), p-IRE1/IRE1 (G), ATF6/GAPDH (H), ATF4/GAPDH (I), and CHOP/GAPDH (J). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, I/R group vs . sham group, one-way analysis of variance followed by Dunnett’s multiple comparison test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; I/R: cerebral ischemia-reperfusion; IRE1: inositol requiring enzyme 1; MCAO: middle cerebral artery occlusion; p-: phosphorylated-; TTC: 2,3,5-triphenyl tetrazolium chloride.

Article Snippet: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China, Cat# C1088) following the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Staining, MANN-WHITNEY, Comparison, Binding Assay

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

Article Snippet: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China, Cat# C1088) following the manufacturer’s instructions.

Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Secreted neuroserpin ameliorates cell death in cultured cortical neurons. (A) Schematic of the experiment: cell lysates and media of cortical neurons (7 DIV) were collected separately and analyzed individually for NSP levels after 4 hours of OGD. (B) Representative western blot images showing the cellular and secreted levels of NSP. (C) Quantifications of NSP protein levels in cell lysate or medium from primary cortical neurons after 4 hours of OGD by Western blot assay ( n = 6). **** P < 0.0001, OGD 4 h vs . control, unpaired t -test. (D) Quantifications of the cellular NSP mRNA level in cell lysate from primary cortical neurons after 4 hours of OGD by quantitative polymerase chain reaction ( n = 7). **** P < 0.0001, OGD 4 h vs . control, unpaired t -test. (E) Neuroserpin mRNA levels in brain tissues of sham and MCAO mice by quantitative polymerase chain reaction ( n = 3 mice per group). ** P < 0.01, control vs . MCAO, unpaired t -test. (F) A diagram showing the conditional medium treatment procedure. Conditioned medium was collected from normal neurons of the same stage (7 DIV) and added to OGD-injured neurons for the reperfusion phase. To neutralize NSP in the medium, anti-NSP antibody (α-NSP Ab; 50 ng for 20,000 cells) was added in the conditional medium. (G) Cell viability was measured by MTT assay in OGD/R neurons cultured in conditioned medium with or without α-NSP Ab ( n = 3). ** P < 0.01, OGD/R vs. control; # P < 0.05, OGD/R + conditioned medium vs . OGD/R; $ P < 0.05, OGD/R + conditioned medium + α-NSP Ab vs. OGD/R + conditioned medium, unpaired t -test. (H) A diagram showing the recombinant NSP treatment procedure. NSP (1−20 ng/mL) was added to neurons 4 hours before OGD and during OGD and after reperfusion. (I) Cell viability of neurons treated with different concentrations of NSP was determined by MTT assay ( n = 4). **** P < 0.0001, OGD/R vs. control; # P < 0.05, OGD/R + NSP vs . OGD/R, unpaired t -test. (J) Representative images of live and dead neurons, treated with 20 ng/mL NSP, measured by the live and death cell assay. Scale bar: 200 µm. (K) Quantification of the percentage of dead cells ( n = 3). *** P < 0.001, OGD/R vs . control; ## P < 0.01, OGD/R + NSP vs. OGD/R, unpaired t -test. (L) A diagram showing the experimental procedure: NSP (50 and 100 ng/mL) was added to neurons immediately after OGD. (M) Cell viability of neurons treated with different concentrations of NSP were determined by MTT assay ( n = 3). **** P < 0.0001, OGD/R vs . control; # P < 0.05, OGD/R + NSP vs . OGD/R, unpaired t -test. (N) Representative TUNEL and DAPI staining in OGD/R-injured neurons treated with NSP (20 and 100 ng/mL). Scale bar: 100 µm. (O) Number of TUNEL-positive cells in each group was quantified and normalized to that of the control group ( n = 3). ** P < 0.01, OGD/R vs. control; ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. α-NSP Ab: Anti-NSP antibody; DAPI: 4′,6-diamidino-2-phenylindole; DIV: day in vitro ; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MCAO: middle cerebral artery occlusion; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China, Cat# C1088) following the manufacturer’s instructions.

Techniques: Cell Culture, Western Blot, Control, Real-time Polymerase Chain Reaction, MTT Assay, Recombinant, TUNEL Assay, Staining, In Vitro

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Neuroserpin inhibits ER stress–mediated signaling transduction induced by OGD/R. (A) Schematic representation of the timing of the experimental procedures. Neuroserpin (NSP; 20 ng/mL) was added to cortical neurons (7 DIV) 4 hours before OGD and during OGD, followed by reperfusion. Cell lysates were collected at the indicated time points of reperfusion for the examination of ER stress signaling molecules. (B) Representative western blots showing phosphorylated and total levels of ER stress sensors and apoptosis-related proteins. (C) Representative western blots showing levels of puromycin. (D–K) Quantitative analysis of the normalized p-PERK/PERK (D, n = 3), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 5), CHOP (J, n = 5), and cleaved-caspase-3 (K, n = 4). GAPDH and α-tubulin were used as loading controls. ** P < 0.01, *** P < 0.001, OGD/R vs . control; # P < 0.05, ## P < 0.01, OGD/R + NSP vs . OGD/R, unpaired t -test. Data are shown as mean ± SEM. ATF: Activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; i.c.v.: intracerebroventricular; NSP: neuroserpin; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase.

Article Snippet: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China, Cat# C1088) following the manufacturer’s instructions.

Techniques: Transduction, Western Blot, Control, Binding Assay, In Vitro

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: Transcriptomic analysis reveals that neuroserpin rescues ER stress and neuronal apoptosis in MCAO mice. (A) PCA of different groups (sham group, n = 5; MCAO group, n = 3; NSP treated group, n = 5). (B) Volcano plots of DEGs between MCAO and sham groups (MCAO vs . sham). (C) Volcano plots of DEGs between NSP and MCAO groups (NSP vs. MCAO). For B and C, gray represents no significant change in expression, red represents upregulation, and blue represents downregulation. The dotted horizontal line represents an adjusted P value of 0.05. The dotted vertical line represents that fold change reaches 2-fold. (D) GO enrichment analysis of DEGs (MCAO vs. sham). (E) GO enrichment analysis of DEGs (NSP vs . MCAO). In D and E, the yellow and blue bars represent the selected enriched GO terms from the upregulated and downregulated DEGs, respectively. Bar length represents the gene ratio. Red numbers are the adjusted P values. (F) The dot plot shows the selected enriched gene sets by GSEA (MCAO vs . sham and NSP vs . MCAO). Dots in blue and red represent downregulated and upregulated gene sets, respectively. The size of dots represents the adjusted P value. (G, H) Venn diagram shows the genes that were reversely regulated by NSP. (G) Upregulated DEGs in MCAO but downregulated DEGs in NSP. (H) GO enrichment analysis of reversed DEGs from G. (I) Downregulated DEGs in MCAO and upregulated DEGs in NSP. (J) GO enrichment analysis of reversed DEGs from I. The color depth of dots represents the adjusted P value. DEGs: Differentially expressed genes; GO: gene ontology; GSEA: Gene Set Enrichment Analysis; MCAO: middle cerebral artery occlusion; NSP: neuroserpin; PCA: principal components analysis.

Article Snippet: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using a One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China, Cat# C1088) following the manufacturer’s instructions.

Techniques: Expressing

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: After PASMCs infected with the corresponding siRNA were treated under normoxic or hypoxic conditions, the cells were incubated with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (HY‐K1079, MCE) for 1 h in a dark environment.

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: After PASMCs infected with the corresponding siRNA were treated under normoxic or hypoxic conditions, the cells were incubated with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (HY‐K1079, MCE) for 1 h in a dark environment.

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: 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: After PASMCs infected with the corresponding siRNA were treated under normoxic or hypoxic conditions, the cells were incubated with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (HY‐K1079, MCE) for 1 h in a dark environment.

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