Journal: iScience

Article Title: In vitro vascularized immunocompetent patient-derived model to test cancer therapies

doi: 10.1016/j.isci.2023.108094

Figure Lengend Snippet:

Article Snippet: Mouse anti-CD31 , Cell Applications , Cat# CB13678.

Techniques: Plasmid Preparation, Recombinant, Lysis, RNA Extraction, Amplification, DC Protein Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Software, Fluorescence, Microscopy

Journal: Materials Today Bio

Article Title: Delivered baicalein immunomodulatory hydrogel with dual properties of pH-responsive and anti-infection orchestrates pro-regenerative response of macrophages for enhanced hypertrophic scars therapy

doi: 10.1016/j.mtbio.2025.102160

Figure Lengend Snippet: Functional performance and biocompatibility evaluation of hydrogels. (A) Photographs of MRSA and E.coil colonies, and (B, C) corresponding quantitative calculation of antibacterial rates after treatments of different hydrogels. (D) Representative scratch assay images of L929 cells treated with different hydrogels and (E) corresponding quantitative analysis of cell migration rates based on scratch area. ELISA was performed to assess the expression of CD31 (F) and VEGF (G) in HUVEC cells following treatment with various hydrogels. (H) Photographs of hemolysis assays and (I) statistically analyzed hemolysis rates. (J) Cytocompatibility of hydrogels and in vivo hematological parameters, including (K)white blood cell (WBC) count -, (L)red blood cell (RBC) count , and (M)platelet count. n = 3, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Following seeding in 12-well culture plates, HUVECs were maintained for 72 h in experimental media supplemented with different test materials, after which the supernatants were collected, centrifuged, and analyzed for CD31 (E-EL-H6227, Elabscience) and VEGF (E-EL-H0111, Elabscience) concentrations.

Techniques: Functional Assay, Wound Healing Assay, Migration, Enzyme-linked Immunosorbent Assay, Expressing, In Vivo

Journal: Nature

Article Title: Capillary cell-type specialization in the alveolus.

doi: 10.1038/s41586-020-2822-7

Figure Lengend Snippet: Fig. 1 | Two stable, intermingled alveolar capillary cell types. a, Alveolar capillaries in adult mouse lung immunostained for PECAM1. b, t-distributed stochastic neighbour embedding (t-SNE) plot of endothelial cell populations annotated in scRNA-seq data for adult mouse lung13. c, Heat map of expression of capillary subset markers and the general endothelial marker Cldn5 in individual capillary cells. CPM, counts per million. d–f, Single-molecule fluorescent in situ hybridization (smFISH) for the capillary subset markers Apln (d, f) or Ednrb and Car4 (aCap) (e), and Aplnr (gCap) (d–f), in adult mouse lung. Images in d (right) and f show individual aCap and gCap cells. g, Relative abundance of aCap cells, gCap cells and cells that co-express aCap and gCap markers (intermediate (IM) cells) in lungs from 3-month-old (young) and 24-month-old (aged) mice (data shown as mean; n = 500 cells scored per mouse; 2 mice per group). h, i, Co-expression of tdTomato lineage label (asterisks) and aCap marker Ednrb but not gCap marker Aplnr (h), or gCap marker Aplnr but not aCap marker Apln (i), in lungs collected six months after mature aCap (h) or gCap (i) cells were lineage-labelled. Blue, DAPI. Scale bars, 10 μm.

Article Snippet: CD34 (BD Biosciences, 347660):https://www.bdbiosciences.com/eu/applications/research/clinical-research/oncology-research/ 3 nature research | reporting sum m ary O ctober 2018 blood-cell-disorders/surface-markers/human/purified-mouse-anti-human-cd34-my10/p/347660 Endomucin (Invitrogen, eBioV.7C7, 14-5851-82): https://www.thermofisher.com/antibody/product/Endomucin-Antibody-cloneeBioV-7C7-V-7C7-Monoclonal/14-5851-82 Integrin alpha8 (R&D, AF4076): https://www.rndsystems.com/products/mouse-rat-integrin-alpha8-antibody_af4076 Pecam1 (rat anti-mouse; BD Biosciences, 553370): https://www.bdbiosciences.com/ds/pm/tds/553370.pdf Pecam1 (mouse anti-human; R&D, BBA7): https://www.rndsystems.com/products/human-cd31-pecam-1-antibody-9g11_bba7 tdTomato (Rockland, 600-401-379): https://rockland-inc.com/store/Antibodies-to-GFP-and-Antibodies-to-RFP-600-401-379O4L_24299.aspx VE-Cadherin (R&D, AF938): https://www.rndsystems.com/products/human-ve-cadherin-antibody_af938 The following primary antibodies were used to stain turtle and/or alligator tissue.

Techniques: Expressing, Marker, In Situ Hybridization

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a The accumulation of platelets and HRG during laser injury-induced thrombus formation in murine cremaster arterioles were visualized using Dylight-649-congugated anti-CD42c (Red) and Alexa-488-conjugated anti-HRG (Green), respectively, with irrelevant IgG as background control (488-IgG) (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity of HRG ( b ) and platelets ( c ) were calculated and plotted over time from mice treated or untreated with eptifibatide (10 μg/g body weight, repeated every 15~20 min). The area under the curve (AUC) for platelets ( d ) and HRG ( e ) were analyzed from each individual thrombus in different treatment groups as indicated. The number of thrombi ( n value) analyzed in each group was indicated above the bars. f A representative focal plane of HRG accumulation and vascular endothelium, as detected by Alexa-488-conjugated anti-HRG (Green) and Alexa-647-conjugated anti-CD31 (Red), respectively, approximately 5 min after laser-induced vessel injury, was extracted from in vivo two-photon scanning of the cremaster arterioles. The areas of the cremaster arteriole (ROI1, cyan) and the thrombus (ROI2, magenta) were manually defined (scale bar: 20 μm). g The intensities of green (HRG) and red (CD31) fluorescence were plotted spatially along the white solid line as indicated in f . h The scatter plot of pixel intensities of green (HRG) and red (CD31) fluorescence in ROI1 and ROI2 as indicated in f were presented. The Pearson’s correlation coefficients (r) for pixels with intensity above the threshold (indicated by the dashed lines) in the thrombus (ROI2) and in the area without the thrombus (ROI1-ROI2) were inserted. Veh vehicle, Epti eptifibatide. Data are presented as mean values ± SEM and analyzed by Kruskal–Wallis test with Dunn’s multiple comparisons ( d and e ). Source data are provided as a Source Data file.

Article Snippet: HRG and CD31 were visualized in the growing thrombus using Alexa-488-conjugated anti-HRG (0.5 μg/g body weight) (Angio-Proteomie) and Alexa-647-conjugated anti-CD31 (0.5 μg/g body weight) (Biolegend), respectively.

Techniques: Control, Fluorescence, In Vivo

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a Mouse plasma pre-treated with recombinant mouse PDI-CCCC or PDI-AAAA was incubated on mouse cerebral microvascular endothelial cells (bEnd.3) in the presence of Zn 2+ (100 μM). The binding of HRG on the cell surface were determined by immunofluorescence using Alexa-488-conjugated antibodies (scale bar: 20 μm). b Representative images of immunohistochemistry of HRG from cross sections of thrombus induced by FeCl in the carotid artery in mice treated with vehicle or Rutin (scale bar: 250 μm). c Representative images of HRG incorporation (Green), visualized using Alexa-488-conjugated anti-HRG, at indicated time points following laser injury in the cremaster arterioles in mice treated with vehicle or Rutin (5 μg/g body weight) (scale bar: 30 μm) (Supplementary Movie 2). The median fluorescence intensity ( d ) and the area under the curve (AUC) ( e ) of HRG were analyzed from each individual thrombus in the two groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. f Mouse plasma pre-treated with recombinant mouse PDI-CCCC or PDI-AAAA was incubated on bEnd.3 cells in the presence of Zn 2+ (100 μM). The binding of antithrombin on the cell surface were determined by immunofluorescence using Alexa-488-conjugated antibodies (scale bar: 20 μm). g Representative images of immunohistochemistry of antithrombin from cross sections of thrombus induced by FeCl in the carotid artery in mice treated with vehicle or Rutin (scale bar: 250 μm). h Representative images of antithrombin incorporation (Green), visualized using Alexa-488-conjugated anti-antithrombin, at indicated time points following laser injury in the cremaster arterioles in mice treated with vehicle or Rutin (5 μg/g body weight) (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity ( i ) and AUC ( j ) of antithrombin were analyzed from each individual thrombus in the two groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. Veh vehicle, Rutin quercetin-3-rutinoside. Data are presented as mean values ± SEM and analyzed by two-tailed Mann–Whitney U -test ( e and j ). Source data are provided as a Source Data file.

Article Snippet: HRG and CD31 were visualized in the growing thrombus using Alexa-488-conjugated anti-HRG (0.5 μg/g body weight) (Angio-Proteomie) and Alexa-647-conjugated anti-CD31 (0.5 μg/g body weight) (Biolegend), respectively.

Techniques: Recombinant, Incubation, Binding Assay, Immunofluorescence, Immunohistochemistry, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a The activity of antithrombin was measured in WT and Hrg −/− plasma on endothelial cell surface, or in the presence or absence of heparin. The number of independent samples (n value) analyzed in each group was indicated above the bars. b Immunoblotting of HRG antigen in equal amounts of plasma from F12 −/− mice treated with control or vivo-siRNA to HRG, with transferrin as the loading control. c Representative images of platelet accumulation (Red) and fibrin generation (Green), as visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively, at indicated time points following laser injury in the cremaster arterioles in F12 −/− mice treated with control or vivo-siRNA to HRG (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity of platelets ( d ) and fibrin ( e ) were calculated and plotted over time. The area under the curve (AUC) for platelets ( f ) and fibrin ( g ) were analyzed from each individual thrombus in the two groups. The number of thrombi ( n value) analyzed in each group was indicated above the bars. Ctrl, control. Data are presented as mean values ± SEM and analyzed by two-tailed Welch’s t -test ( a ) or two-tailed Mann–Whitney U -test ( f and g ). Source data are provided as a Source Data file.

Article Snippet: HRG and CD31 were visualized in the growing thrombus using Alexa-488-conjugated anti-HRG (0.5 μg/g body weight) (Angio-Proteomie) and Alexa-647-conjugated anti-CD31 (0.5 μg/g body weight) (Biolegend), respectively.

Techniques: Activity Assay, Western Blot, Control, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a Immunoblotting of HRG and FXII antigens in equal amounts of plasma from 4 groups of mice as indicated: WT , Hrg −/− , F12 −/− and DKO , with transferrin as the loading control. b Tissue factor-induced thrombin generation (TGA) in the plasma from 4 groups of mice was evaluated in the presence of endothelial cells. The peak height ( c ) and area under the curve (AUC) ( d ) from individual TGA curve were calculated in these groups as indicated. The number of animals (n value) analyzed in each group was indicated above the bars. e The time to complete arterial occlusion following FeCl 3 -induced carotid injury were recorded in 4 groups of mice as indicated. The number of animals (n value) analyzed in each group was indicated above the bars. f Representative images of platelet accumulation (Red) and fibrin generation (Green), visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively, at indicated time points following laser injury in the cremaster arterioles in 4 groups of mice as indicated (scale bar: 30 μm) (Supplementary Movie ). The median fluorescence intensity of platelets ( g ) and fibrin ( h ) were calculated and plotted over time. The AUC for platelets ( i ) and fibrin ( j ) were analyzed from each individual thrombus in different groups. The number of thrombi ( n value) analyzed in each group was indicated above the bars. Data are presented as mean values ± SEM and analyzed by Welch’s ANOVA test ( c – e ) or Kruskal–Wallis test with Dunn’s multiple comparisons ( i and j ). Source data are provided as a Source Data file.

Article Snippet: HRG and CD31 were visualized in the growing thrombus using Alexa-488-conjugated anti-HRG (0.5 μg/g body weight) (Angio-Proteomie) and Alexa-647-conjugated anti-CD31 (0.5 μg/g body weight) (Biolegend), respectively.

Techniques: Western Blot, Control, Fluorescence

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: a The disulfide pairs were predicted on the domain structure of mouse HRG based on that of human HRG, with red color indicating the targets of PDI. The gain-of-function mutant of mouse HRG ( gof-HRG) was generated by replacing 6 Cys of the 3 target disulfide bonds of PDI: C246-C261, C270–C467 and C315–C320 to Ala, which locked the variant in a reduced form. The binding of wt-HRG and gof-HRG on heparin ( b ) ( n = 12 independent samples) and FXIIa ( c ) ( n = 9 independent samples) was determined by ELISA. d , e The accumulation of HRG following laser injury in the cremaster arterioles of Hrg −/− mice infused with different HRG variants were visualized using Alexa-488-conjugated anti-HRG. The median fluorescence intensity of HRG ( d ) was plotted over time. The area under the curve (AUC) for HRG ( e ) was analyzed from each individual thrombus in different groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. f – i Platelet accumulation and fibrin generation following laser injury in the cremaster arterioles of Hrg −/− mice infused with different HRG variants were visualized by Dylight-649-congugated anti-CD42c and Alexa-488-conjugated 59D8 antibody, respectively. The median fluorescence intensity of platelets ( f ) and fibrin ( g ) were calculated and plotted over time. The AUC for platelets ( h ) and fibrin ( i ) were analyzed from each individual thrombus in different groups. The number of thrombi (n value) analyzed in each group was indicated above the bars. Data are presented as mean values ± SEM and analyzed by two-tailed Welch’s t -test ( b and c ) or two-tailed Mann–Whitney U -test ( e , h and i ). Source data are provided as a Source Data file.

Article Snippet: HRG and CD31 were visualized in the growing thrombus using Alexa-488-conjugated anti-HRG (0.5 μg/g body weight) (Angio-Proteomie) and Alexa-647-conjugated anti-CD31 (0.5 μg/g body weight) (Biolegend), respectively.

Techniques: Mutagenesis, Generated, Variant Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis

doi: 10.1038/s41467-024-47493-0

Figure Lengend Snippet: 1 . Under physiological condition, antithrombin binds to vascular heparan sulfate (HS) to inhibit the activation of coagulation; 2 . Vessel injury induces the release of Zn 2+ and PDI from activated platelets and endothelial cells; 3 . Extracellular PDI cleaves allosteric disulfide bonds in plasma HRG; 4 . Reduced HRG by PDI binds to endothelial HS to displace antithrombin, promoting rapid activation of coagulation. 5 . Reduced HRG by PDI also binds to FXIIa during thrombus growth, preventing excessive clot formation.

Article Snippet: HRG and CD31 were visualized in the growing thrombus using Alexa-488-conjugated anti-HRG (0.5 μg/g body weight) (Angio-Proteomie) and Alexa-647-conjugated anti-CD31 (0.5 μg/g body weight) (Biolegend), respectively.

Techniques: Activation Assay, Coagulation