Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 7. Validation of compound-target interactions through molecular docking and protein-small molecule binding assays. (A) Chemical structure of EGCG and molecular docking models showing its interactions with NOX2 and p47phox. (B) Chemical structure of quercetin and molecular docking models illustrating its interactions with NOX2 and p47phox. (C) An SPR sensorgram displaying the binding affinity of the NOX2-EGCG interaction. (D) An SPR sensorgram showing the binding affinity of the NOX2-quercetin interaction. (E) A BLI sensorgram illustrating the binding affinity of the p47phox-EGCG interaction. (F)A BLI sensorgram depicting the binding affinity of the p47phox-quercetin interaction. Abbreviations: BLI, Bio-layer interferometry; EGCG, epigallocatechin-3-gallate; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; Que, quercetin; SPR, surface plasmon resonance.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: Biomarker Discovery, Binding Assay, SPR Assay

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 8. QKPC and its components, EGCG and quercetin, reduce NOX2-p47phox/ROS signaling in vivo and in vitro. (A) Protein levels of NOX2 and p47phox in human neutrophils were assessed by Western blotting analysis, with relative density quantification shown as the ratio of NOX2 or p47phox to GAPDH (n = 3). (B) Protein levels of NOX2 and p47phox in lung tissues of mice were determined by Western blotting analysis and presented as the ratio of NOX2 or p47phox to GAPDH (n = 3). (C) Cellular ROS levels were measured using the oxidant-sensing probe DCFH-DA, and fluorescence intensity was recorded for each group. EGCG, quercetin, and the positive drugs NAC (20 μM, a ROS scavenger), DPI (50 μM, a general NADPH oxidase inhibitor), and GSK (50 μM, a NOX2-specific inhibitor) effectively suppressed ROS levels elevated by the combined CSE and LPS exposure (n = 6–8). (D) MDA levels in lung tissues were measured with an MDA assay kit. Data are presented as mean ± SD, with statistical significance set at p < 0.05. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extract; DPI, Diphenyleneiodonium chloride; EGCG, epigallocatechin-3-gallate; GSK, GSK2795039; LPS, lipopolysaccharide; MDA, malondialdehyde; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; Que, quercetin; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques: In Vivo, In Vitro, Western Blot, Fluorescence, Multiple Displacement Amplification

Journal: Phytomedicine

Article Title: Qingke Pingchuan granules alleviate airway inflammation in COPD exacerbation by inhibiting neutrophil extracellular traps in mice

doi: 10.1016/j.phymed.2024.156283

Figure Lengend Snippet: Fig. 9. Proposed mechanisms of QKPC action against ECOPD. Abbreviations: COPD, chronic obstructive pulmonary disease; EGCG, epigallocatechin-3-gallate; LPS, lipopolysaccharide; MPO, myeloperoxidase; NE, neutrophil Elastase; NET, neutrophil extracellular trap; NOX2, NADPH oxidase 2; p47phox (NCF1), 47 kDa neutrophil oxidase factor; QKPC, Qingke Pingchuan; ROS, reactive oxygen species.

Article Snippet: Recombinant human NOX2 protein was acquired from CUSABIO (Wuhan, China), while recombinant human P47phox protein was sourced from TargetMol Co., Ltd. (USA).

Techniques:

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (A) or CCL2 (B) at concentrations of 5, 10, 50, or 100 ng/mL, and cell viability was determined by cell counting using a hemocytometer or MTT assay, respectively. (C) Flow cytometry results are presented as histograms of the average percentage of cells that expressed IGF-1R and CCR2 receptors (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 4/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to the control group: p < 0.05 (*) or p < 0.0001(***); significant value compared to control group and the other treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Cell Counting, MTT Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by fluorescence microscopy. ( A ) Photomicrographs show the expression of FN ascertained by immunofluorescence and fluorescence microscopy analysis. Magnification: 400× ( B ) Bars correspond to the quantitative analysis of FN expression in tEnd.1 cells in selected microscopic fields (n = 5/group). The results are expressed in pixels/μm 2 . ( C ) Flow cytometry results are presented as histograms of the average percentage of cells that expressed CD49e/VLA-5 and CD44 receptors for FN (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 5/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.0001 (***); significant values compared to control group and single treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: (A) tEnd.1 cells treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h on BSA or FN coating were stained with Alexa 488-phalloidin and analyzed by confocal microscopy with a 63× objective. ( B ) tEnd.1 cells were allowed to adhere on BSA- or FN-coated surfaces for 1 h after stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 24 h. ( C ) tEnd.1 cells were allowed to migrate through transwell chambers coated with BSA or FN after chemotactic stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 6 h. Photomicrographs demonstrate cells invading through the transwell membrane. Giemsa staining. Scale bar = 10 μm. ( D ) Bars represent the number of migrating cells in a transwell system. Data are represented as mean ± SEM (n = 5/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (***); significant values compared to control group and the IGF-1 treatment: p < 0.05 (#); and significant values compared to control group and single treatments: p < 0.01 (+).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Staining, Confocal Microscopy, Membrane, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: ( A ) tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by optical microscopy. Photomicrographs show intracellular lumina in tEnd.1 cells, indicated by arrows. Giemsa staining. Scale bar = 10 μm. ( B ) tEnd.1 cells were treated with IGF-1, CCL2, or IGF-1/CCL2 for 8 days on BSA or FN coating and analyzed by optical microscopy. Photomicrographs demonstrate capillary-like structures, indicated by asterisks. Giemsa staining. Scale bar = 10 μm. ( C ) Number of capillary-like structures. ( D ) Luminal area of capillary-like structures. Bars represent the mean ± SEM (n = 6/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant compared with control, p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Microscopy, Staining, Control

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: XCL1 siRNA treatment abolished anti-apoptotic effect of human Wharton's jelly-derived human mesenchymal stem cell (WJ-MSC). (a) Before coculturing with serum-starved C2C12 cells, WJ-MSCs were separately pretreated with two siRNAs of human XCL1 containing different sequences for 24 hours. Each siRNA-treated WJ-MSC was cocultured with serum-deprived C2C12 cells for 12 hours. Human-recombinant XCL1 was added to cocultured C2C12 cells with siRNA-treated WJ-MSC to monitor gain of function of XCL1. C2C12 cell images were taken at each condition. (b) Cell lysates were analyzed by Western blot using anti-poly ADP-ribose polymerase (PARP) antibody. Cleaved PARP fragments were monitored by densitometric analysis (*P < 0.05, n = 3). (c) Secreted XCL1 concentration in each conditioned media was measured by XCL1 ELISA kit (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Derivative Assay, Recombinant, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Induction of human XCL1 reduces apoptosis of the skeletal muscles in the zebrafish myopathy model. Twenty hours post fertilization (hpf) zebrafish embryos (a–d) and 36 hpf embryos were observed (e–h). (a,e) The apoptotic cells of control and adssl1 morphants or human XCL1-induced adssl1 morphants were detected by TUNEL assay, and the whole-mounted embryos were observed by fluorescent microscopy. (b,f) The boxed region of the embryos was photographed using confocal microscopy. (c,g) The representative confocal images show that the number of dead cells in adssl1 morphants was more than in control morphants, and induction of human XCL1 inhibited the increase of cell death in adssl1 morphants. (d,h) The cell death in the selected area (within five somites in the trunk) was quantified and presented in the scattered chart. A, anterior; P, posterior. a,e, bar = 100 µm; b,c,f,g, bar = 50 µm. d,h, Student's t-test: *P < 0.05, **P < 0.005, ***P < 0.001.

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Muscles, Control, TUNEL Assay, Microscopy, Confocal Microscopy

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Identification of XCL1 as a paracrine factor of Wharton's jelly-derived human mesenchymal stem cell (WJ-MSC) during coculture. Secreted proteins in the media collected from the experiments were measured using the RayBio Biotin Label-based Human Antibody. (a,b) Increased fold of spot intensity of XCL1, IL-3 R, and smad5 in cocultured cells as compared to WJ-MSC alone. (c) The conditioned media was collected from C2C12, WJ-MSC alone, and cocultured cells. Concentration of secreted XCL1 in each conditioned media was measured by XCL1 ELISA kit (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Recombinant XCL1 protein inhibited serum starvation-induced apoptosis of C2C12 cells. C2C12 cell was serum-deprived during culture both in the absence or presence of recombinant XCL1 proteins (1, 10, and 20 ng/ml) for 12 hours. (a) Fluorescence-activated cell-sorting (FACS) analysis of cells stained with annexin V/7-AAD. (b) Percentage of apoptotic cells by FACS analysis (*P < 0.05, n = 3). (c) Harvested cells were analyzed by Western blot analysis with anti-poly ADP-ribose polymerase (PARP) antibody. (d) PARP cleaved bands were analyzed by densitometry (*P < 0.05, n = 3). (e) Serum-deprived C2C12 cells were treated with recombinant XCL1 (8 ng/ml) in a time-dependent manner. (f) PARP cleavage was monitored by Western blot analysis (*P < 0.05, n = 3). (g) Serum-deprived C2C12 cells were treated with either PAN caspase inhibitor (z-VAD-FMK) or XCL1 protein in a dose-dependent manner for 24 hours. (h) Apoptotic cells were analyzed by Western blot with anti-PARP antibody. (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Recombinant, Fluorescence, FACS, Staining, Western Blot

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: Absence of anti-apoptotic effect of XCL1 in other types of cells. The HT22, mouse hippocampal neuron cells were treated with amyloid-β42 (1 μmol/l for 48 hours) or MG132 (5 μmol/l for 24 hours) in the absence or presence of XCL1. (a) Fluorescence-activated cell-sorting (FACS) analysis of cells stained with annexin V/7-AAD. (b) Poly ADP-ribose polymerase (PARP)-cleaved bands were analyzed by densitometry. S16 rat Schwann cells were treated with thapsigargin (0.1 μmol/l for 12 hours) in the absence or presence of XCL1. The thapsigargin (0.1 μmol/l for 12 hours) treated S16 cells were analyzed by (c) FACS analysis and (d) Western blot analysis with anti-PARP antibody.

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Fluorescence, FACS, Staining, Western Blot

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: XCL1 treatment inhibited lovastatin-induced apoptosis of C2C12 cells and reduction of myotube formation. (a) C2C12 cells were treated with lovastatin (0.1, 0.5, 1, and 2 μmol/l) in a dose-dependent manner in the absence or presence of XCL1 and protein lysates were analyzed by Western blot analysis using anti-poly ADP-ribose polymerase (PARP) antibody. (b) Cleaved PARP fragments were monitored by densitometric analysis (*P < 0.05, n = 3). (c) The 7-day differentiated myotubes from C2C12 cells were treated with lovastatin (1, 2.5, 5, and 10 μmol/l) in the absence or presence of XCL1. Each arrow indicates defects in the myotubes. (d,e) Protein extracts of myotubes were analyzed by Western blot analysis using anti-myosin heavy chain (MHC) antibody. Bands of MHC were monitored by densitometric analysis (*P < 0.05, n = 3).

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Western Blot

Journal: Molecular Therapy

Article Title: Anti-apoptotic Effects of Human Wharton's Jelly-derived Mesenchymal Stem Cells on Skeletal Muscle Cells Mediated via Secretion of XCL1

doi: 10.1038/mt.2016.125

Figure Lengend Snippet: The rescue of defective skeletal muscles by human XCL1 in the zebrafish model. (a) The boxed region indicates the observed area to determine the effect of human XCL1 on skeletal muscles in zebrafish at 36 hpf. Myosepta and muscle fibers were stained with anti-Laminin and myosin heavy chain (MHC) antibodies, respectively. (b) The whole-mounted embryos after double staining with anti-Laminin and MHC antibodies were observed by confocal microscopy. The adssl1 morphants (phosphate-buffered saline injected embryos) showed disrupted muscle fibers and myosepta, whereas the morphants injected with human XCL1 showed recovery of muscle defects. (c) The zebrafish embryos demonstrating skeletal muscle phenotype were counted and quantified data were presented graphically. The data indicates that human XCL1 restores muscle abnormality of adssl1 morphants in a dose-dependent manner. A, anterior; P, posterior. Scale bar = 50 µm.

Article Snippet: The C2C12 myoblasts (1 × 10 5 cells/well of a six-well plate) were treated with or without human recombinant XCL1 (0–5 nmol/l) (R&D Systems, Minneapolis, MN) for indicated time periods in serum-free medium.

Techniques: Muscles, Staining, Double Staining, Confocal Microscopy, Saline, Injection

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Specific recognition of hyaluronan by the Ig-like-V-set domain of hSiglec-9. (a) Immobilized high molecular weight-hyaluronan (HMW-HA) was probed with human Siglec–Fc and CD44–Fc chimeras and binding evaluated by using an anti-human IgG-HRP. (b) Binding of hSiglec-9–Fc to immobilized HMW-HA was performed in the presence of increasing concentrations of HMW-HA, heparan sulfate, chondroitin sulfate or heparin. (c, d) To map the hSiglec-9 domain responsible for HA recognition, binding of hSiglec-9–Fc to immobilized HMW-HA was compared to binding of hSiglec-9–Fc with an Arg→Ala mutation in the V-set domain (hSiglec-9R120K), a fusion protein construct of the hSiglec-9 V-set domain + the second Ig-like domain (C2-set) of hSiglec-7 + human IgG Fc tail (hSiglec-9V-7C2–Fc), a fusion protein construct encompassing V-set domain of hSiglec-7 + C2-set of hSiglec-9 + human IgG Fc tail (Siglec-7V-9C2–Fc), a fusion protein construct of the hSiglec-9 V-set domain + first C2-set domain + human IgG Fc tail (Siglec-9–Fc 2D). Results are expressed as the mean ± SD. All experiments were performed in triplicate, repeated 3 times (a, d) or 2 times (b). One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Mutagenesis, Construct, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: High molecular weight hyaluronan (HMW-HA) is recognized by hSiglec-9 on human neutrophils. (a) To evaluate the molecular size of hyaluronan (HA) responsible for binding to hSiglec-9, high molecular weigh HA (HMW-HA; >1,000 kDa), low molecular weigh (200 – 300 kDa-HA and 25 – 75 kDa-HA), nano-HA (nonamers) and oligo-HA (hexamers) fragments were added to compete with binding of hSiglec-9–Fc to immobilized HMW-HA plates. Binding was evaluated using an anti-human IgG-HRP. Experiment performed in triplicate and repeated 3 times; results are expressed as mean ± SD. (b) Flow cytometry reveals constitutive expression of Siglec-9 and CD44 on human neutrophils from 9 different donors; geometric mean ± 95% confidence interval. (c) Binding of FITC-labeled HMW-HA to neutrophils of these donors was evaluated by flow cytometry; geometric mean ± 95% confidence interval. (d) Human neutrophils were pretreated with anti-Siglec-9 monoclonal antibodies, anti-human CDw329 (BD Pharmingen, #550906) and anti-human Siglec-9 (R&D Systems, #BAF1139) and effects on binding to FITC-labeled HMW-HA determined. CDw329 Ab blocked binding of HMW-HA, but not GBS capsule (Fig. 5b) and was designated “α-Sig-9(HA)”; in contrast, the R&D Systems Ab blocked recognition of GBS capsule (Fig. 5b), but did not interfere with binding to HMW-HA, and was thus designated “α-Sig-9(Sia)”. Data pooled from five independent experiments in triplicate; data represent the mean ± SD. (e, f) Calcein-labeled human neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) and α-CD44 mAbs, added to wells coated with immobilized HMW-HA to facilitate adherence, then unbound neutrophils washed away. Remaining neutrophils were lifted and enumerated. Experiment performed in triplicate and repeated 5 times; results are expressed as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Flow Cytometry, Expressing, Labeling, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) engages hSiglec-9 via its surface hyaluronan capsule. (a) Human Siglec-9–Fc was immobilized to ELISA wells using protein A, and binding of FITC-labeled forms of WT GAS, its isogenic HA capsule-deficient mutant (ΔhasA), an animal-passaged hyperencapsulated (AP) derivative and its isogenic HA capsule-deficient mutant (AP ΔhasA) were evaluated; results are expressed as mean ± SD and repeated 5 times in triplicate with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) (b) Human Siglec-9–Fc chimera was immobilized to ELISA wells via protein A in the presence of α-Sig-9(HA), α-Sig-9(Sia) or isotype control Abs and binding of FITC-labeled WT GAS or sialic acid-expressing serotype III group B Streptococcus (GBS) evaluated. (c) Human Siglec-9/9R120K/5/6/7/11 and human CD44-Fc chimera were immobilized to ELISA wells via protein A. Binding of FITC-labeled WT GAS was evaluated. Results represent mean ± SD; triplicate wells, representative experiment depicted of 5 independent repeats with similar results, performed in triplicate. (d) hSiglec-9–Fc was immobilized to ELISA plates using protein A, then wells were pretreated with HMW-HA, HMW-heparan sulfate or heparin over the indicated range of concentrations. Binding of FITC-labeled WT GAS was evaluated.

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Mutagenesis, Comparison, Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: HMW-HA binding to hSiglec-9 induces SHP-1 recruitment and blunts neutrophil NET production and oxidative burst. (a) 1.2 × 107 human neutrophils were incubated for 30 min + 10 µg/ml of high molecular weight hyaluronan (HMW-HA) + 25 nM PMA. Cell lysates were immunoprecipitated with α-Siglec9 and SHP-1 recruitment was visualized by western blot analysis; results were repeated 2 times with similar results; representative experiment with relative densitometry values is shown. (b) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 Abs, incubated with 10 µg/ml of HMW-HA and activated for 30 min with PMA. Reactive oxygen species (ROS) release was measured with the OxyBURST Green H2HFF BSA probe and results expressed as mean fluorescence intensity (MFI) ± SD; experiment repeated 5 times with similar results; representative experiment is shown. (c) Neutrophils were pretreated α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs, then incubated with 10 µg/ml of HMW-HA and activated with PMA for 3 h; production of neutrophil extracellular traps (NETs) visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); representative fields at 20x magnification is shown; experiment performed in triplicate and repeated 5 times. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD; experiment repeated 3 times in triplicate with similar results; representative experiment shown. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Incubation, Molecular Weight, Immunoprecipitation, Western Blot, Fluorescence, Staining, Picogreen Assay, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) binding to hSiglec-9 via its surface HMW-HA capsule blunts neutrophil oxidative burst, NET responses and bactericidal activity. (a, b) Neutrophils were labeled with OxyBURST Green H2HFF BSA in the presence of α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at MOI = 20 for 30 min and oxidative burst measured by FACS; results are expressed as MFI ±SD and repeated twice with similar results; representative experiment is shown. (c) PMA-stimulated neutrophils (5 × 105 cells) were pretreated with α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs and exposed for 3 h to MOI = 10 of GAS (DNase mutant) that had been pretreated or not with hyaluronidase to remove HA capsule and NET production visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); results are repeated 5 times in triplicate, representative fields at 32 × magnification is shown. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD and repeated 2 times with similar results; representative experiment shown. (e) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at multiplicity of infection (MOI) = 10 for 30 min, then cells lysed and dilutions plated on agar for enumeration of colony forming units to evaluate neutrophil killing of GAS. Data represent the mean + SD of triplicates; repeated 4 times with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) or P < 0.05 (*).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Activity Assay, Labeling, Infection, Mutagenesis, Staining, Picogreen Assay, Comparison

Journal: Nature Communications

Article Title: Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions

doi: 10.1038/s41467-022-29855-8

Figure Lengend Snippet: a – d Upon blue light irradiation, Vasa-mCherry-LOV was recruited to actin, resulting in Vasa-less micromeres at the 16-cell stage in the experimental group. A level of translation was detected by FIAsH-EDT 2 at the 16-cell stage and was increased in the micromere-side of the spindle in the control group ( a and b ) whereas decreased in that of the experimental group ( c and d ). A whole Z-section of the cell of interest (0.44 μm per slice; ~90 slices in total) was imaged and analyzed every 1-min and presented as a maximum 2D-projection. A bright green spot (*) is a non-specific debris that tends to fluoresce under the microscope. Each experiment was repeated at least three times and the measurement results of two other embryos are shown in Supplementary Fig. . e , f Localization of β-catenin-GFP was tracked by time-lapse imaging of every 15 min for ~5 h after activation (approximately, ~12 hpf). In controls, GFP-β-catenin enriched into the vegetal most cells ( e , arrows), whereas its localization became uniform or random in the experimental groups ( f , arrows). Arrowheads indicate ectopic localization of GFP-β-catenin. A whole Z-section of the embryo of interest (1 μm per slice; ~50 slices in total) was imaged and is presented as a maximum 2D-projection. The embryos were lightly squashed to completely immobilize during imaging. Therefore, the embryos that successfully formed micromeres under this condition were selected for the analysis and the embryos in a similar angle were compared between control and experimental groups in this study. These experiments were performed at least three independent times. All Scale bars = 10 μm. Source data are provided as a Source Data file.

Article Snippet: Similarly, Dendra2 (Addgene #54694) was subcloned into pSP64-vector and GFP-Spβ-catenin (Logan and McClay, 1999; SPU_009155) was synthesized by IDT (Illinois, USA) and inserted into the same vector as described above. pCS2-2xmCherry-EMTB and pCS2-EMTB-3xGFP were obtained at Addgene (#26741). pSP64-SpNanos-GFP was previously made .

Techniques: Irradiation, Microscopy, Imaging, Activation Assay

Journal: Nature Communications

Article Title: Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions

doi: 10.1038/s41467-022-29855-8

Figure Lengend Snippet: A list of all materials used in this study.

Article Snippet: Similarly, Dendra2 (Addgene #54694) was subcloned into pSP64-vector and GFP-Spβ-catenin (Logan and McClay, 1999; SPU_009155) was synthesized by IDT (Illinois, USA) and inserted into the same vector as described above. pCS2-2xmCherry-EMTB and pCS2-EMTB-3xGFP were obtained at Addgene (#26741). pSP64-SpNanos-GFP was previously made .

Techniques: Recombinant, Labeling, Clone Assay, Plasmid Preparation, Sequencing, Software, Imaging

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca 2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse / rat / human Substance P (rabbit polyclonal), Cy3 , Bioss USA , Cat # bs-0064R-Cy3.

Techniques: Functional Assay, Recombinant, Staining, Avidin-Biotin Assay, Blocking Assay, Plasmid Preparation, Inhibition, Fluorescence, SYBR Green Assay, Activation Assay, Bicinchoninic Acid Protein Assay, In Situ, Enzyme-linked Immunosorbent Assay, Activity Assay, Labeling, Software

Journal: Scientific Reports

Article Title: Defective membrane repair machinery impairs survival of invasive cancer cells

doi: 10.1038/s41598-020-77902-5

Figure Lengend Snippet: Expression and subcellular localization of endogenous Anx in MDA-MB-231. ( a ) 10 µg of protein extracts from MDA-MB-231 and BeWo cells were analyzed by western-blot. The gel analysis plugin of ImageJ was used to quantify the density of bands. The histogram presents mean values (± SD) of the ratio Anx/GAPDH from ten independent experiments. Student t Test for independent samples. ** p < 0.01. ( b ) Revelation of AnxA5 in MDA-MB-231 and BeWo cells compared to 50 ng purified recombinant AnxA5 (Pure A5), presented as an example. The whole membrane, together with representative western-blot analysis for AnxA1, A2, A4 and A6, are presented in Supplementary Fig. . ( c ) MDA-MB-231 cells were immunostained for AnxA1, AnxA2, AnxA4, AnxA5 and AnxA6 (green), as indicated, and counterstained with DAPI (blue). The right-hand column presents magnified images extracted from the “Anx” column (indicated by inserts). Scale bar: 40 µm. ( d ) Quantification of fluorescence intensities after immunostaining of endogenous Anx in MDA-MB-231 cells. Fluorescence intensities were measured from 8-bit images by drawing a circular ROI inside the cytoplasm and measuring the mean pixel value. Mean pixel values from at least 20 cells are presented. Horizontal bars represent median values. The variance value is 323, 268, 224, 2372 and 2909 for AnxA1, A2, A4, A5 and A6, respectively. The coefficient of variation is 14%, 13%, 24%, 35% and 47% for AnxA1, A2, A4, A5 and A6, respectively. The expression level of AnxA5 or AnxA6 is heterogenous from one MDA-MB-231 cell to another. In some cells, AnxA5 and AnxA6 are expressed at a very low level.

Article Snippet: Semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content in AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA4 (32 kDa), AnxA5 (35 kDa), AnxA6 (68 kDa), and glyceraldehyde-3-phosphate deshydrogenase (GAPDH, loading control, 37 kDa) was detected with rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA4 monoclonal antibody (SAB4200121, Sigma), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-AnxA6 monoclonal antibody (sc-271859, Santa cruz Biotechnology), and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Expressing, Western Blot, Purification, Recombinant, Membrane, Fluorescence, Immunostaining

Journal: Stroke

Article Title: Neuronal CD200 Signaling Is Protective in the Acute Phase of Ischemic Stroke.

doi: 10.1161/STROKEAHA.120.032374

Figure Lengend Snippet: Figure 1. Effects of neuronal CD200 signaling on stroke outcomes and neuronal apoptosis at 3 days after stroke. (A) Representative images of brain slices stained with cresyl violet (CV) and quantification of brain infarct volumes, NDS, and corner test scores in TMX vs. VEH mice. (B) TUNEL labelled cell apoptosis in peri-infarct area of TMX and VEH treated mice and the quantification of percent of TUNEL positive cells in ipsilateral hemispheres Each dot represents the average of % apoptotic cells from eight 20× fields/animal in the peri-infarct area. N=3 for sham and 6–7 for stroke per group; *P < 0.05.

Article Snippet: Determination of CD200 and cytokine Levels The levels of CD200 in mouse brain, plasma and in the supernatants of primary neuronal culture were measured by a sandwich ELISA kit according to manufacture instruction ((# EK1185, Mouse CD200 PicoKine ELISA Kit, Boster Biological, Pleasanton, CA, USA.).

Techniques: Staining, TUNEL Assay

Journal: Stroke

Article Title: Neuronal CD200 Signaling Is Protective in the Acute Phase of Ischemic Stroke.

doi: 10.1161/STROKEAHA.120.032374

Figure Lengend Snippet: Figure 4. Plasma cytokine levels after ischemic injury. Pro-inflammatory (TNFα, IL-1β and IL-6) and anti-inflammatory (IL-10 and IL-4) cytokine levels (pg/mL) were measured in the plasma of VEH and TMX treated sham/stroke mice at 3d (A) and 7d (B). N=5–6 sham and 6–7 stroke animals per group; *P < 0.05. (C) CD200 levels in naïve brains (left), in the plasma (middle) of TMX and VEH treated mice, and in neuronal culture medium after OGD (right). N=5–6 sham and 6–8 stroke animals per group; *P < 0.05. For neuronal culture medium, N=4 independent OGD experiments; *P < 0.05.

Article Snippet: Determination of CD200 and cytokine Levels The levels of CD200 in mouse brain, plasma and in the supernatants of primary neuronal culture were measured by a sandwich ELISA kit according to manufacture instruction ((# EK1185, Mouse CD200 PicoKine ELISA Kit, Boster Biological, Pleasanton, CA, USA.).

Techniques: Clinical Proteomics

Journal: Stroke

Article Title: Neuronal CD200 Signaling Is Protective in the Acute Phase of Ischemic Stroke.

doi: 10.1161/STROKEAHA.120.032374

Figure Lengend Snippet: Figure 5. Stroke outcomes and flow cytometry in CD200 Fc treated mice. (A) Representative images of brain slices stained with cresyl violet (CV) and quantification of brain infarct volumes, NDS, and corner test scores in CD200Fc vs. IgG treated mice. N=3 for sham and 6–7 for stroke per group; *P < 0.05. (B) Microglial expression of cell membrane markers CD68/CD206 measured as mean fluorescence intensity (MFI). (C) Microglial expression of intracellular markers IL-1β/TNFα. For (B) and (C), n=4/sham group and n=6/stroke group; *P < 0.05.

Article Snippet: Determination of CD200 and cytokine Levels The levels of CD200 in mouse brain, plasma and in the supernatants of primary neuronal culture were measured by a sandwich ELISA kit according to manufacture instruction ((# EK1185, Mouse CD200 PicoKine ELISA Kit, Boster Biological, Pleasanton, CA, USA.).

Techniques: Flow Cytometry, Staining, Expressing, Membrane, Fluorescence

Journal: Stroke

Article Title: Neuronal CD200 Signaling Is Protective in the Acute Phase of Ischemic Stroke.

doi: 10.1161/STROKEAHA.120.032374

Figure Lengend Snippet: Figure 6. Interaction of neuronal CD200 with lymphocytic CD200R1. (A) The purity of CD45+CD3+

Article Snippet: Determination of CD200 and cytokine Levels The levels of CD200 in mouse brain, plasma and in the supernatants of primary neuronal culture were measured by a sandwich ELISA kit according to manufacture instruction ((# EK1185, Mouse CD200 PicoKine ELISA Kit, Boster Biological, Pleasanton, CA, USA.).

Techniques: