Journal: Nature Communications

Article Title: The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity

doi: 10.1038/s41467-024-45303-1

Figure Lengend Snippet: Antibodies

Article Snippet: Sections were blocked with 5% normal donkey serum (Vector Laboratories)/0.1%Triton-X/0.01 M PBS for 1 h and incubated with polyclonal goat anti-mouse aminopeptidase N/CD13 for pericyte coverage.

Techniques:

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: (A) Flow-cytometry dot-plots demonstrating predominant FITC-fXII binding for PS-positive platelet subpopulations (labelled “PS+”). The result corresponds to 1000 nM FITC-fXII binding. (B) The effect of platelets on activation of purified 200 nM fXII. Platelets were activated by 10 nM thrombin (n = 3). (C) Dose-dependence for platelet-dependent fXIIa formation upon a reaction between thrombin-activated platelets and purified fXII (n = 3). A hyperbolic curve for a representative, typical experiment was fit using K m fXII = 64 and [ fXIIa ] max = 0.126 nM. The given K m and [ fXIIa ] m values are mean values (±SD) calculated using data from three independent experiments. (D) The effect of different platelet stimulation methods on platelet-dependent fXIIa formation in 20% plasma. The asterisks symbols (*) correspond to p<0.05. represents the p values for comparing fXII activating capacities between activated and non-activated platelets.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Flow Cytometry, Binding Assay, Activation Assay, Purification, Clinical Proteomics

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: Stimulation and platelet-dependent fXII activation.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Activation Assay, Significance Assay

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: (A) Computational model schematic for fXII activation on the platelet surface. Soluble fXII first binds the surface and is then activated; fXIIa can inhibit further activation through an auto-inhibition mechanism . The reaction rate constants ( k i ) for the model were estimated based on the data in and , and the surface area of an activated platelet was considered 50 μm 2 . (B) Platelet-dependent fXIIa formation in buffer as a function of the fXII concentration added. The black circles correspond to the experimental data (±SD for three independent experiments, n = 3); the curve corresponds to the computational model. The consistency between the data and model curve confirms the fXII auto-inhibition hypothesis. (C) The dose-dependence of fXIIa formation in 20% plasma for A23187-activated platelets after platelet secretion removal. The platelets were activated at 2×10 8 /mL and diluted to the indicated concentrations for further reaction with plasma. The black squares represent the experimental data (n = 3), and the curve represents the computational model. The consistency between the data and model curve confirms the reliability of the model.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Activation Assay, Inhibition, Concentration Assay, Clinical Proteomics

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: (A) The effect of 20 μg/mL annexin V or 1.5 μM prothrombin on 280 nM fXII activation (in the presence of 2.5 mM CaCl 2 ) by A23187-activated (secretion-depleted) platelets (n = 3). (B) Comparison of the fXIIa-generating capacities for A23187-activated platelets (at 2×10 6 /mL) and 4 μM PS- or PC-liposomes (depicted as PS and PC, respectively) in 20% chelated plasma. The mean values (±SD) were calculated from three independent experiments (n = 3). (C) Confocal microscopy analysis of poly-P localisation in activated platelets with 2.5 mM calcium. DAPI was used as poly-P marker , and AlexaFluor-647-annexin V was used as a marker for procoagulant PS-positive platelets (labelled “PS+”). PS-negative activated platelets are labelled “PS-” (n = 3). (D) Confocal microscopy analysis of poly-P localisation in activated platelets without 2.5 mM calcium. FITC-anti-fibrin(ogen) antibody was used as an activated platelet marker (n = 3). The images in (C) and (D) were collected at an ∼2 μm z-depth over the fibrinogen surface to reduce non-specific fibrinogen immunofluorescence.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Activation Assay, Comparison, Liposomes, Clinical Proteomics, Confocal Microscopy, Marker, Immunofluorescence

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: (A) The effect of platelet secretion removal on platelet-related fXII activation (means±SD for three independent experiments, n = 3). A portion of the A23187-activated platelet suspension was diluted to 4×10 6 /mL and mixed 1:1 with 40% chelated plasma to measure the initial fXIIa-generating capacity in the presence of platelet secretion (which was estimated at 100%). The remaining platelets were washed free from platelet secretion, diluted, and analysed. (B) The effect of platelet secretion on 2 nM fXIIa activity (n = 3). The reaction in buffer A without platelets was compared with the reaction in the presence of 2×10 6 /mL activated platelets or platelet secretion (at an equivalent dilution). (C) Western blot analysis of purified C1-INH (labelled “C1-INH”) and platelet secretion (labelled “Secretion”). Supernatant containing platelet secretion was incubated with 25 nM fXIIa, and the total protein was concentrated 20-fold. After resolution using 4–10% SDS-PAGE gels and subsequent Western blotting, the bands were developed using the ECL method. The figures on the left depict the molecular masses of markers measured in kDa (n = 4).

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Activation Assay, Suspension, Clinical Proteomics, Activity Assay, Western Blot, Purification, Incubation, SDS Page

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: ( A ) We used a scheme that depicts the platelet aggregate to test the C1-INH washout hypothesis. The platelet concentration inside the thrombi was considered 1 per 15 fl. The initial C1-inhibitor distribution in the aggregate for the simulations is shown in grey, and at values ranging from 0.1 to 10 μm/s, the flow penetrated the aggregate. At t = 0, C1-INH at 100 μM (estimated from ref. ) and fXII at 450 nM appeared simultaneously. Factor XII could be activated on the platelet surface, and C1-INH could diffuse through the aggregate (D = 10 μm 2 /s, based on the molecular weight) and move due to the flow. The fXIIa diffusion was assumed negligible because fXII activation is surface-associated ( and this study), and typically, fXIIa is tightly bound to the activation surface . The C1-INH action was described using a mass action equation with the reaction constant 0.00366 μM -1 s -1 . ( B ) Time-course of the distance-averaged [C1-INH] for various flow velocities. ( C ) Time-course of the surface-averaged [fXIIa] for various flow velocities. The C1-INH and fXIIa spatial distributions were governed by a set of differential equations, which were solved using the finite volume solver available within the Virtual Cell environment.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Concentration Assay, Molecular Weight, Diffusion-based Assay, Activation Assay

Journal: The European journal of neuroscience

Article Title: VEGF-E Attenuates Injury After Ischemic Stroke by Promoting Reparative Revascularization.

doi: 10.1111/ejn.70114

Figure Lengend Snippet: FIGURE 3 | Revascularization is stabilized by VEGF-E associated with attenuation of neuronal degeneration. (a) Representative fluorescence images of CD31 and PDGFRβ immunolabeling at the injury site in vehicle (VEH)- and VEGF-E-treated mice 4 days after stroke. (b) Analysis of the density of perivascular PDGFRβ+ cells at the injury site 4 days after stroke. (c) Analysis of CD31 and PDGFRβ colocalization at the injury site. (d) Analysis of the number of CD31+ microvascular stalls at the injury site. (e) Representative fluorescence images of CD13 and CD105 immunolabeling at the injury site in VEH- and VEGF-E-treated mice. (f) Analysis of the density of CD105+ microvessels at the injury site. (g) Analysis of CD105 and CD13 colocalization at the injury site. (h) Representative fluorescence images of FJB+ degenerating neurons in the ipsilateral cortex and ipsilateral striatum. Stereological analysis of the density of FJB+ degenerating neurons in the (i) ipsilateral cortex and (j) ipsilateral striatum. Data are boxplot with min/max or stacked histogram (n = 6–7 animals/group). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control (b, d, f, i, unpaired two-tailed t- test). Abbreviations: IF, immunofluorescence; VEGF, vascular endothelial growth factor; VEH, vehicle.

Article Snippet: The following primary antibodies were used: rat anti- mouse cluster of differentiation (CD31) (1/500; BD Biosciences, ON, Canada; 550274), goat anti- mouse aminopeptidase N (CD13) (1/250; R&D systems), rat anti- mouse glial fibrillary acidic protein (GFAP) (1/500; Invitrogen, MA, USA; 13- 0300), rabbit anti- mouse ionized calcium binding adaptor molecule (IBA1) (1/500; WAKO, ON, Canada; 019- 19741), goat anti- rat CD45 (1/500; BD Biosciences; 553076), rabbit anti- mouse PDGFRβ (1/250; Abcam, ON, Canada; ab32570), rat anti- mouse endoglin (CD105) (1/250; Novus Biologicals, ON, Canada; NB100- 77666), and rabbit anti- mouse aquaporine- 4 (AQP4) (1/500; Cell Signaling Technology, MA, USA; 59678S).

Techniques: Fluorescence, Immunolabeling, Control, Two Tailed Test, Immunofluorescence

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Pericyte number and coverage is reduced along blood–spinal cord barrier. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 2-month-old wild-type mouse cortex, caudate, and hippocampal brain regions and cervical, thoracic, and lumbar spinal cord regions (anterior horns). ( B ) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive brain and spinal cord capillaries. Mean±s.e.m., n =5 to 7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( C ) Quantification of regional CD13-positive pericyte cell number normalized to collagen IV-positive capillary surface area in the brain and spinal cord. Mean±s.e.m., n =5 to 7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( D ) Confocal microscopy analysis of platelet-derived growth factor receptor β (PDGFR β )-positive pericytes (green) and lectin capillary profiles (red) in 2-month-old wild-type mouse cortex, caudate, and hippocampal brain regions and cervical, thoracic, and lumbar spinal cord regions. ( E ) Quantification of regional PDGFR β -positive pericyte coverage of lectin-positive brain and spinal cord capillaries. Mean±s.e.m., n =7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05. ( F ) Quantification of regional PDGFR β -positive pericyte cell number normalized to lectin-positive capillary surface area in the brain and spinal cord. Mean±s.e.m., n =7 animals per group. # P <0.05 when compared with the brain regions; * P <0.05.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Confocal Microscopy, Derivative Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Subregional variation in spinal cord pericyte number and coverage between anterior horn gray matter and lateral and dorsal funiculi. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 2-month-old wild-type mouse anterior horn gray matter or dorsal columns for cervical thoracic and lumbar spinal cord. ( B ) Quantification of CD13-positive pericyte coverage of collagen IV-positive capillaries in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =5 to 7 animals per group; * P <0.05. ( C ) Quantification of CD13-positive pericyte cell number normalized to lectin-positive capillary surface area in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =5 to 7 animals per group; * P <0.05. ( D ) Quantification of platelet-derived growth factor receptor β (PDGFR β )-positive pericyte coverage of lectin-positive capillaries in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =7 animals per group; * P <0.05. ( E ) Quantification of PDGFR β -positive pericyte cell number normalized to lectin-positive capillary surface area in anterior horn gray matter (gray) or dorsal and lateral funiculi (white) from cervical, thoracic, and lumbar spinal cord. Mean±s.e.m., n =7 animals per group; * P <0.05.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Confocal Microscopy, Derivative Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Blood–spinal cord barrier pericyte reductions contribute to increased capillary permeability

doi: 10.1038/jcbfm.2012.113

Figure Lengend Snippet: Exacerbation of pericyte deficiency leads to overt blood–spinal cord barrier disruption and leakage of endogenous plasma proteins. ( A ) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 6-month-old Pdgfrβ +/+ and Pdgfrβ F7/F7 mouse cervical, thoracic, and lumbar spinal cord anterior horn. ( B ) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive anterior horn spinal cord capillaries. Mean±s.e.m., n =3 animals per group; * P <0.05. ( C ) Representative confocal microscopy analysis of IgG (green), fibrin (red), and lectin-positive capillaries (blue) in 6-month-old Pdgfrβ +/+ mouse lumbar anterior horn and anterior horn from Pdgfrβ F7/F7 cervical, thoracic, and lumbar spinal cord. ( D , E ) Quantification of IgG ( D ) and fibrin ( E ) extravascular deposits in the spinal cord regions in tissue sections from 2-month-old B6SJL/F1 Pdgfrβ +/+ , 6-month-old Pdgfrβ +/+ 129S1/SvlmJ, and 6-month-old Pdgfrβ F7/F7 mice. Mean±s.e.m., n =3 animals per group; * P <0.05. ( F , G ) Confocal microscopy analysis of thrombin (white) ( F ) or fibrin (red) ( G ) and SMI-311-positive neurons (blue) in 6-month-old Pdgfrβ +/+ and Pdgfrβ F7/F7 lumbar spinal cord. Arrows indicate neuronal accumulation.

Article Snippet: Sections were then incubated in the following primary antibodies: goat α -mouse CD13 (10 μ g/mL, R&D Systems), mouse α -mouse desmin (1:100, Dako, Carpinteria, CA, USA), rabbit α -mouse collagen IV (1:200, EMD Millipore), rabbit α -human fibrin which cross reacts with mouse fibrin (1:500, Dako), goat α -mouse thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse α -mouse zonula occludens-1 (ZO-1) (1:50, Invitrogen), mouse α -mouse occludin (1:50, Invitrogen), mouse α -mouse neuronal-specific antigen A60 (NeuN) (1:100, EMD Millipore) and goat anti-mouse ChAT (choline acetyl transferase) (1:50, EMD Millipore).

Techniques: Disruption, Clinical Proteomics, Confocal Microscopy

Journal: Frontiers in Oncology

Article Title: YBX1 Enhances Metastasis and Stemness by Transcriptionally Regulating MUC1 in Lung Adenocarcinoma

doi: 10.3389/fonc.2021.702491

Figure Lengend Snippet: The expression levels of YBX1 affected lung adenocarcinoma cells migration, invasion, and stemness. (A, B) The wound healing assay analyzed cell migration in A549 and H1299 cells, and YBX1 expression affected the migration rate. (C) YBX1 expression affected the EMT signaling and MMPs family. (D) The transwell assay without matrigel analyzed cell migration in A549 and H1299, and YBX1 expression affected the migrated cells number. (E) The transwell assay with matrigel analyzed cell invasion in A549 and H1299, and YBX1 expression affected the invaded cells number. (F) The sphere formation analyzed cancer cell stemness in A549 and H1299 cells. The number and diameter of the spheres were affected by YBX1 expression. (G) The markers related to stemness were detected by western blot, including CD44, CD133, Oct-4. (H) ALDH1 positive population analysis revealed that decreased expression of YBX1 reduced the ALDH1+ cell proportions, whereas overexpression of YBX1 increased the proportion. The data are presented as mean ± SD of three independent tests. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The antibodies against GAPDH (10494-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), CD133 (14553-1-AP), MUC1 (19976-1-AP), and CD44 (15675-1-AP) and the goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (HAF008) and goat-anti-mouse IgG conjugated to HRP (HAF007) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Migration, Wound Healing Assay, Transwell Assay, Western Blot, Over Expression

Journal: Frontiers in Oncology

Article Title: YBX1 Enhances Metastasis and Stemness by Transcriptionally Regulating MUC1 in Lung Adenocarcinoma

doi: 10.3389/fonc.2021.702491

Figure Lengend Snippet: MUC1 overexpression partly rescued the down-regulation of metastasis and stemness caused by YBX1 silencing. (A) Cell migration was analyzed by wound healing assay in A549, and the migration rate was partly rescued by MUC1 overexpression. (B) Cell migration was analyzed by transwell assay without matrigel, and the migrated cells were partly rescued by MUC1 overexpression. (C) Cell invasion was analyzed by transwell assay with matrigel, and the invaded cells were partly rescued by MUC1 overexpression. (D) MUC1 overexpression partly rescued the regulation of the EMT signaling and MMPs family caused by YBX1 silencing. (E) Cancer cell stemness was analyzed by sphere formation in A549. The number and diameter of the spheres were partly rescued by MUC1 overexpression. (F) ALDH1 positive population analysis revealed that MUC1 overexpression increased the number of ALDH1+ cells reduced by YBX1 silencing. (G) MUC1 overexpression partly rescued the regulation of the markers related to stemness, including CD44, CD133, Oct-4, caused by YBX1 silencing. The data are presented as mean ± SD of three independent tests. *P < 0.05, **P < 0.01.

Article Snippet: The antibodies against GAPDH (10494-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), CD133 (14553-1-AP), MUC1 (19976-1-AP), and CD44 (15675-1-AP) and the goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (HAF008) and goat-anti-mouse IgG conjugated to HRP (HAF007) were purchased from Proteintech (Wuhan, China).

Techniques: Over Expression, Migration, Wound Healing Assay, Transwell Assay

Journal: Frontiers in Oncology

Article Title: YBX1 Enhances Metastasis and Stemness by Transcriptionally Regulating MUC1 in Lung Adenocarcinoma

doi: 10.3389/fonc.2021.702491

Figure Lengend Snippet: YBX1 promotes tumor growth by upregulating MUC1 in human lung cancer xenograft mouse models. (A) The morphology of all mice participating in the experiment was photographed. (B) The morphology of tumor xenografts from all mice was photographed. (C, D) The tumor volume and weight from the different groups were measured. (E) The tumor volume of each mouse was measured at a regular interval of 3 days after 7 days of injection. (F) The expression of YBX1, MUC1, CD133, Oct-4, MMP9, E-cadherin, β-catenin, and Snail within xenografts in the different groups were detected by Western blot. (G) The expression of YBX1, MUC1, CD133, E-cadherin, and Snail in tumor tissues was detected by IHC staining. The xenograft tissue morphology showed by HE staining. Scale bars= 50μm. The level of significance was indicated by **P < 0.01

Article Snippet: The antibodies against GAPDH (10494-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), CD133 (14553-1-AP), MUC1 (19976-1-AP), and CD44 (15675-1-AP) and the goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (HAF008) and goat-anti-mouse IgG conjugated to HRP (HAF007) were purchased from Proteintech (Wuhan, China).

Techniques: Injection, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Frontiers in Oncology

Article Title: YBX1 Enhances Metastasis and Stemness by Transcriptionally Regulating MUC1 in Lung Adenocarcinoma

doi: 10.3389/fonc.2021.702491

Figure Lengend Snippet: YBX1 promotes tumor metastasis by upregulating MUC1 in lung cancer metastasis models. (A) The GFP-labeled A549 from different groups were injected into the tail vein of nude mice, and in vivo imaging was performed 45 days later. The lungs are dissected and imaged individually. (B) All whole lung was fixed with 4% paraformaldehyde, and the surface of the fixed metastases was white granular. (C) The number of metastases in the lungs of each nude mouse was counted. (D) The expression of YBX1, MUC1, CD133, E-cadherin, β-catenin, and Snail within xenografts in the different groups was detected by Western blot. (E) The lung adenocarcinoma metastasis morphology showed by HE staining. Scale bars= 500μm. N= 3 mice/group. Original magnification of the whole lung: ×1.25.

Article Snippet: The antibodies against GAPDH (10494-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), CD133 (14553-1-AP), MUC1 (19976-1-AP), and CD44 (15675-1-AP) and the goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (HAF008) and goat-anti-mouse IgG conjugated to HRP (HAF007) were purchased from Proteintech (Wuhan, China).

Techniques: Labeling, Injection, In Vivo Imaging, Expressing, Western Blot, Staining