recombinant murine high molecular weight upa  (Innovative Research Inc)

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: Trafficking of neutrophils (N), classical monocytes (cMOs), non‐classical monocytes (ncMOs), B lymphocytes, CD4 + T lymphocytes, and CD8 + T lymphocytes to the peritoneal cavity as assessed 6 h after intraperitoneal stimulation with recombinant murine uPA, tPA, PAI‐1, uPA‐PAI‐1, or tPA‐PAI‐1 in WT mice by multi‐channel flow cytometry. The gating strategy and quantitative data are shown (mean ± SEM for n = 5 mice per group; # P < 0.05 vs. saline; one‐way ANOVA). Neutrophil and cMO trafficking to the peritoneal cavity elicited by recombinant murine uPA‐PAI‐1 as assessed in WT mice receiving neutrophil‐depleting anti‐Ly‐6G mABs or isotype control ABs, quantitative data are shown (mean ± SEM for n = 4 mice per group; # P < 0.05 vs. saline; * P < 0.05 vs. isotype; one‐way ANOVA). Intravascular endothelial cell interactions and transmigration of neutrophils (Ly‐6G + CX 3 CR‐1 − ; red) and cMOs (Ly‐6G − CX 3 CR‐1 low ; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI‐1, or uPA‐PAI‐1 in postcapillary venules of the cremaster of CX 3 CR‐1 GFP/+ mice by multi‐channel in vivo microscopy. Representative still images (scale bar: 50 µm) and quantitative data are shown (mean ± SEM for n = 4 mice per group; # P < 0.05 vs. saline; * P < 0.05 vs. uPA; § P < 0.05 vs. PAI‐1; one‐way ANOVA).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Recombinant, Flow Cytometry, Transmigration Assay, In Vivo, Microscopy

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: A Deposition of uPA or PAI‐1 (green) in the cremaster muscle of WT mice as assessed separately ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of TNF or saline, PECAM‐1/CD31 + postcapillary venules (red) and Ly‐6G + neutrophils (purple) are depicted. Representative images are shown (scale bar: 50 µm). B Expression of PECAM‐1/CD31 (red) and ICAM‐1/CD54 or VCAM‐1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA‐PAI‐1 or saline, representative images (scale bar: 50 µm), and quantitative data are shown (mean ± SEM for n = 4 mice per group; # P < 0.05 vs. saline; t ‐test). C, D (C) Production of TNF was analyzed in mouse RAW 264.7 macrophages and (D) surface expression of ICAM‐1/CD54, VCAM‐1/CD106, uPA, and PAI‐1 on mouse bEnd.3 microvascular endothelial cells as assessed in vitro by multi‐channel flow cytometry upon exposure to recombinant murine uPA‐PAI‐1, TNF, or saline, quantitative data are shown (mean ± SEM for n = 4 experiments per group; # P < 0.05 vs. saline; one‐way ANOVA/ t ‐test). E, F (E) Binding of ICAM‐1/CD54‐Fc or VCAM‐1/CD106‐Fc to primary mouse neutrophils and (F) binding of conformation‐specific mABs to primary human neutrophils (“mAB 24” recognizing high‐affinity conformation, “kim127” recognizing intermediate and high‐affinity conformation of β 2 integrins) as assessed upon exposure to recombinant murine/human uPA‐PAI‐1, TNF, or saline by multi‐channel flow cytometry, a representative histogram plot and quantitative data are shown (mean ± SEM for n = 4–7 mice/human blood samples per group; # P < 0.05 vs. saline; one‐way ANOVA). G Quantitative analysis of LFA‐1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E‐selectin/CD62E + ICAM‐1/CD54, E‐selectin/CD62E + ICAM‐1/CD54 + KC, or E‐selectin/CD62E + ICAM‐1/CD54 + KC + uPA‐PAI‐1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 µm; mean ± SD; n = 3 mice; n = 10–13 flow chambers, n = 122–222 cells); * P < 0.05 vs. E‐selectin/CD62E + ICAM‐1/CD54; one‐way ANOVA).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Ex Vivo, Confocal Laser Scanning Microscopy, Injection, Expressing, Recombinant, In Vitro, Flow Cytometry, Binding Assay, Confocal Microscopy

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: Production of different cytokines by mouse RAW macrophages upon exposure to recombinant murine uPA‐PAI‐1 heteromers or saline as assessed by multiplex ELISA, results are shown as heatmap. In addition, quantitative data are provided for the most abundantly produced cytokines (mean ± SEM for n = 5 experiments per group; # P < 0.05 vs. saline; t ‐test).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Recombinant, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Produced

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: A, B uPA‐PAI‐1‐elicited recruitment of neutrophils (N) and classical monocytes (cMOs) to the peritoneal cavity of WT mice treated with anti‐LFA‐1/CD11a, anti‐Mac‐1/CD11b, anti‐VLA‐4/CD49d, anti‐ICAM‐1/CD54, anti‐VCAM‐1/CD106 mABs (A), anti‐VLDLr mABs, anti‐LRP‐1 Abs, the MAPK inhibitors FR180204 (ERK1/2), SB202580 (p38), SP600125 (JNK) (B), or isotype control antibodies/drug vehicle as assessed by multi‐channel flow cytometry, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; # P < 0.05 vs. saline; * P < 0.05 vs. isotype/vehicle; one‐way ANOVA). C Recruitment of neutrophils (N) and classical monocytes (cMOs) to the peritoneal cavity of WT mice treated with recombinant murine uPA‐PAI‐1, recombinant human uPA‐recombinant murine PAI‐1 heteromers, recombinant murine DFP‐uPA‐PAI‐1, or vehicle (mean ± SEM for n = 4–6 mice per group; # P < 0.05 vs. saline; one‐way ANOVA).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Flow Cytometry, Recombinant

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: A Correlation of uPA or PAI‐1 protein expression and neutrophil infiltration in tumors as assessed by ELISA as well as immunohistochemistry and light microscopy in human breast cancer samples (histological grade: G1), representative images (scale bar: 100 µm) and quantitative data are shown. B Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI‐1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cutoff z ≥ 2.0) in the METABRIC breast cancer cohort. C Surface expression of MMP‐9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA‐PAI‐1 (uPA‐PAI‐1) by multi‐channel flow cytometry, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; # P < 0.05 vs. saline; t ‐test). D, E Proliferation of (D) 4T1 breast cancer cells or (E) bEnd.3 microvascular endothelial cells upon exposure to recombinant murine uPA‐PAI‐1, the uPA‐PAI‐1 inhibitor WX‐340, or primary neutrophils isolated from the peritoneal cavity of WT mice undergoing 6 h of intraperitoneal stimulation with uPA‐PAI‐1 with or without addition of a NE inhibitor as assessed by a MTT assay, quantitative data are shown (mean ± SEM for n = 3 experiments per group; # P < 0.05 vs. neutrophils; * P < 0.05 vs. neutrophils + vehicle / # P < 0.05 vs. 2 % FCS medium; one‐way ANOVA/ t ‐test). F Formation of NETs (histone H3 + ; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intrascrotal injection of uPA‐PAI‐1, TNF, or saline, PECAM‐1/CD31 + postcapillary venules (blue) and Ly‐6G + neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Light Microscopy, RNA Expression, Marker, Isolation, Flow Cytometry, Recombinant, MTT Assay, Ex Vivo, Confocal Microscopy, Injection

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: A, B Correlation of uPA or PAI‐1 protein expression (ELISA) and neutrophil infiltration (histochemistry and light microscopy) in human breast cancer samples, representative images (A; histological grades: G1‐3; scale bar: 100 µm) and quantitative data (B; histological grades: G2 or G3; n = 10–29 samples per group).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Light Microscopy

Journal: EMBO Molecular Medicine

Article Title: uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

doi: 10.15252/emmm.202013110

Figure Lengend Snippet: Effect of compound WX‐340 on binding of recombinant murine uPA to PAI‐1 protein as assessed by ELISA, quantitative data are shown (mean ± SEM for n = 3 experiments per group; # P < 0.05 vs. drug vehicle; one‐way ANOVA). Intravascular rolling and firm adherence as well as transmigration of neutrophils and classical monocytes (cMOs) to the inflamed perivascular tissue as assessed 6 h after intrascrotal injection of TNF in postcapillary venules of the cremaster muscle of WT mice, quantitative data are shown (mean ± SEM for n = 6 mice per group; # P < 0.05 vs. saline; * P < 0.05 vs. drug vehicle; one‐way ANOVA). Expression of uPA or PAI‐1 (green) in tumors as assessed in an orthotopic model of 4T1 breast cancer in WT mice by confocal microscopy, PECAM‐1/CD31 + postcapillary venules (blue) and Ly‐6G + neutrophils (red) are depicted (scale bar: 100 µm). Relative development rates and tumor weight in animals treated with WX‐340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean ± SEM for n = 4–6 mice per group; * P < 0.05 vs. drug vehicle; one‐way ANOVA). Intravascular rolling and firm adherence of neutrophils as assessed in the tumor microvasculature 14 days after intradermal injection of 4T1 cells in the right ear of WT mice treated with WX‐340 or vehicle by multi‐channel in vivo microscopy, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; * P < 0.05 vs. drug vehicle; t ‐test). Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX‐340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi‐channel flow cytometry, quantitative data are shown (mean ± SEM for n = 4–6 mice per group; * P < 0.05 vs. drug vehicle; t ‐test).

Article Snippet: Recombinant murine high‐molecular‐weight uPA and PAI‐1 as well as human uPA and PAI‐1 (varying doses; Molecular Innovations, Novi, MI) were used to induce leukocyte responses in different in vitro and in vivo assays (see below).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Transmigration Assay, Injection, Expressing, Confocal Microscopy, In Vivo, Microscopy, Flow Cytometry

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Urokinase-type plasminogen activator (uPA) protects the tripartite synapse in the ischemic brain via ezrin-mediated formation of peripheral astrocytic processes

doi: 10.1177/0271678X18783653

Figure Lengend Snippet: Effect of treatment with recombinant uPA on the formation of peripheral astrocytic processes in the ischemic brain. (A and B) Representative confocal micrographs taken with a 60× lens and electronically magnified 4.7 times of ezrin (white in a, c, e, and g; and red in b, d, f, and h) and synaptophysin (green) expression (A), and mean number of ezrin/synaptophysin (SYP)-positive puncta (B) per 2100 µm2 of tissue in the left frontoparietal cortex of Wt mice (n = 3 per group) after 30 min of tMCAo (panels c, d, g, and h) or sham operation (panels a, b, e, and f) followed by intravenous treatment with ruPA (e and f) or saline solution (SS; c and d). (C) Mean number of intact synaptic contacts denoted by their immunoreactivity to synaptophysin and PSD-95 in the left frontoparietal cortex of Wt mice (n = 3 per group) following 30 min of tMCAo and treatment with SS or ruPA, alone or in the presence of the GS inhibitor (GS-i) L-methionine sulfoximine, or with the inhibitor alone. (D and E) Representative confocal micrographs taken with a 60× lens and electronically magnified 4.7 times of GLT-1 (red) and synaptophysin (green) expression (D), and mean number of GLT-1/synaptophysin crossings (E) per 2100 µm2 of tissue in the left frontoparietal cortex of Wt mice (n = 40 observations per group for four different animals) subjected to the experimental conditions described above. GFAP: glial fibrillary acidic protein; tMCAo: transient occlusion of the middle cerebral artery; GS: glutamine synthetase; ruPA: recombinant urokinase-type plasminogen activator.

Article Snippet: Recombinant murine uPA (ruPA) and uPA’s amino terminal fragment (ATF) were purchased from Molecular Innovations (Novi, MI).

Techniques: Recombinant, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Urokinase-type plasminogen activator (uPA) promotes ezrin-mediated reorganization of the synaptic cytoskeleton in the ischemic brain

doi: 10.1074/jbc.RA118.002534

Figure Lengend Snippet: uPA promotes ezrin-mediated synaptic recovery in the ischemic brain. A, representative confocal microscopy pictures at 40× magnification from dendrites from WT neurons maintained under normoxic conditions (panel a) or 24 h after 5 min of OGD and treatment with either PBS (panel b) or 5 nm of uPA either immediately after OGD (IA; panel c) or 24 h later (panel d). Green is bassoon, and red is PSD-95. B, mean number of synaptic contacts in WT neurons exposed to the experimental conditions described in A. n = 66–74 extensions examined per experimental group from neurons from three different cultures. The lines denote S.E. C, mean number of intact synaptic contacts in WT neurons treated with ezrin siRNA or Sc-siRNA and exposed to 5 min of OGD conditions. Twenty-four hours later, the cells were treated with 5 nm of uPA or as comparable volume of PBS. n = 40 neurons from three different cultures per experimental group. The lines denote S.E. D and E, representative confocal microscopy pictures at 60× magnification and electronically enlarged 4.6 times (D) from the ischemic area (panels a and c) and a comparable zone in the contralateral nonischemic hemisphere (panels b and d), and quantification of number of intact synaptic contacts in each area (E), in WT mice following 30 min of tMCAo and 24 h of recovery. The animals were intravenously treated with either SS or ruPA 24 h after tMCAo. Three hours later, the brains were harvested for the different observations. n = 14 cuts examined per animal from three different animals per experimental group. The lines in E denote S.E.

Article Snippet: Murine ruPA and uPA's N-terminal fragments (ATF) were purchased from Molecular Innovations (Novi, MI).

Techniques: Confocal Microscopy