82 research outputs found

    Enhanced ionomycin induced eryptosis and adhesion of erythrocytes from annexin7-deficient mice. A.

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    <p>Histogram of annexin V-binding reflecting phosphatidylserine exposure in a representative experiment of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>) and their wild type control mice (<i>anx7<sup>+/+</sup></i>) exposed for 30 min to Ca<sup>2+</sup> ionophore ionomycin (1 µM). <b>B.</b> Arithmetic means ± SEM (n = 8−9) of the percentage of annexin V-binding erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) exposed for 30 min to Ringer without (left bars) or with (right bars) ionomycin (1 µM). ### significant (p<0.001) difference from absence of ionomycin, *** significant difference (p<0.001) from <i>anx7<sup>+/+</sup></i> erythrocytes (ANOVA). <b>C.</b> Arithmetic means ± SEM (n = 8−9) of the forward scatter of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) exposed for 30 min to Ringer without (left bars) or with (right bars) ionomycin (1 µM). ### significant (p<0.001) difference from absence of ionomycin, *** significant difference (p<0.001) from <i>anx7<sup>+/+</sup></i> erythrocytes (ANOVA). <b>D.</b> Arithmetic means ± SEM (n = 6) of the number of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) adhering to the human umbilical vein endothelial cells (HUVEC) following exposure for 30 min to Ringer without (left bars) or with (right bars) ionomycin (1 µM). ### significant difference (p<0.001) from absence of ionomycin, *** significant difference (p<0.001) from <i>anx7<sup>+/+</sup></i> erythrocytes (ANOVA).</p

    Role of CXCL16 in dynamic adhesion erythrocytes to anti-CXCl16-treated endothelial cells under arterial shear stress. A.

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    <p>Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow. The erythorcytes were pretreated for 30 minutes with Ringer solution without (control) or with 1 µM ionomycin. The HUVEC were left untreated or treated for 2 hours with neutralizing antibody directed against endothelial CXCL16 (4 µg/ml), ***(p<0.001) indicates statistically significant difference from absence of ionomycin (1 µM), ###(p<0.001) indicates statistically significant difference from anti CXCL 16 (ANOVA). <b>B.</b> Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow. The erythrocytes were pretreated for 12 hours with Ringer with (control), or without (-Glucose) glucose. The HUVEC were left untreated or treated for 2 hours with neutralizing antibody directed against endothelial CXCL16 (4 µg/ml), ***(p<0.001) indicates statistically significant difference from absence of glucose, ###(p<0.001) indicates statistically significant difference from anti CXCL 16 (ANOVA). C. Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow. The erythrocytes were pretreated for 2 hours with isotonic (control) or hypertonic (550 mM sucrose) Ringer. The HUVEC were left untreated or treated for 2 hours with neutralizing antibody directed against endothelial CXCL16 (4 µg/ml), ***(p<0.001) indicates statistically significant difference from absence of hyperosmotic shock, ###(p<0.001) indicates statistically significant difference from anti CXCL 16 (ANOVA).</p

    Enhanced eryptosis and adhesion of erythrocytes from annexin7-deficient mice following osmotic shock. A.

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    <p>Histogram of annexin V-binding reflecting phosphatidylserine exposure in a representative experiment of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>) and their wild type control mice (<i>anx7<sup>+/+</sup></i>) exposed for 2 hours to hyperosmotic shock (550 mM sucrose added). <b>B.</b> Arithmetic means ± SEM (n = 8−9) of the percentage of annexin V-binding erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) exposed for 2 hours to isotonic (left bars) or hyperosmotic (550 mM sucrose added, right bars) Ringer. ### significant (p<0.001) difference from isotonic Ringer, *** significant difference (p<0.001) from <i>anx7<sup>+/+</sup></i> erythrocytes (ANOVA). <b>C.</b> Arithmetic means ± SEM (n = 8−9) of the forward scatter of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) exposed for 2 hours to isotonic (left bars) or hyperosmotic (550 mM sucrose added, right bars) Ringer. ### significant (p<0.001) difference from isotonic Ringer, *** significant difference (p<0.001) from <i>anx7<sup>+/+</sup></i> erythrocytes (ANOVA). <b>D.</b> Arithmetic means ± SEM (n = 6) of the number of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) adhering to human umbilical vein endothelial cells (HUVEC) following exposure of the erythrocytes for 2 hours to isotonic (left bars) or hyperosmotic (550 mM sucrose added, right bars) Ringer. ### significant (p<0.001) difference from isotonic Ringer, *** significant difference (p<0.001) from <i>anx7<sup>+/+</sup></i> erythrocytes (ANOVA).</p

    Annexin-binding erythrocytes in ischemic kidneys from wild type and from annexin deficient mice. A.

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    <p>Arithmetic means ± SEM (n = 7) of erythrocyte abundance in the inner stripe of renal tissue following renal ischemia/reperfusion of anx7<sup>−/−</sup> mice (black bar) and <i>anx7<sup>+/+</sup></i> mice (white bar). <b>B.</b> Arithmetic means ± SEM (n = 7) of the acute tubular necrosis (ATN) score in the inner stripe of renal tissue following renal ischemia of anx7<sup>−/−</sup> mice and <i>anx7<sup>+/+</sup></i> mice, ***(p<0.001) indicate significant difference to wild type mice (ANOVA). <b>C.</b> Arithmetic means ± SEM (n = 7) of the acute tubular necrosis (ATN) score in the outer cortex of renal tissue following renal ischemia of anx7<sup>−/−</sup> mice and <i>anx7<sup>+/+</sup></i> mice, ***(p<0.001) indicate significant difference to wild type mice (ANOVA). <b>D, E.</b> Representative light microscopy image from kidney section of ischemic <i>anx7<sup>+/+</sup></i> mice (<b>D</b>) and anx7<sup>−/−</sup> mice (<b>E</b>) stained with sirius red. Arrows indicate yellow stained erythrocytes within renal capillaries. <b>F, G.</b> Representative light microscopy image from kidney section of ischemic <i>anx7<sup>+/+</sup></i> mice (<b>F</b>) and anx7<sup>−/−</sup> mice (<b>G</b>) stained with PAS.</p

    Role of phosphatidylserine exposure in dynamic adhesion of annexin-treated erythrocytes to endothelial cells under arterial shear stress. A.

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    <p>Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow following exposure for 30 minutes to Ringer solution without (control) or with 1 µM ionomycin without or with a prior 30 min treatment with Annexin V (5 µl/ml). ***(p<0.001) indicates statistically significant difference from absence of ionomycin, ###(p<0.001) indicates statistically significant difference from absence of annexin V (ANOVA). <b>B.</b> Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow following a 12 hours treatment with Ringer solution with (control), or without (-Glucose) glucose without or with a prior 30 min treatment with Annexin V (5 µl/ml). ***(p<0.001) indicates statistically significant difference from absence of glucose, ###(p<0.001) indicates statistically significant difference from absence of annexin V (ANOVA). C. Arithmetic means ± SEM (n = 6) of erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, black bars) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, white bars) binding to human umbilical vein endothelial cells (HUVEC) under flow following a 2 hours exposure to isotonic (control) or hypertonic (550 mM sucrose) Ringer solution without or with a prior 30 min treatment with Annexin V (5 µl/ml). ***(p<0.001) indicates statistically significant difference from absence of hyperosmotic shock, ###(p<0.001) indicates statistically significant difference from absence of annexin V (ANOVA).</p

    Correlation between phosphatidylserine exposure and dynamic adhesion of erythrocytes to endothelial cells under arterial shear stress.

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    <p>Arithmetic means ± SEM (n = 6−8) of the erythrocytes adhering to HUVEC as a function of the percentage erythrocytes binding annexin V. The erythrocytes from annexin7-deficient mice (<i>anx7<sup>−/−</sup></i>, closed symbols) and their wild type control mice (<i>anx7<sup>+/+</sup></i>, open symbols) were left without pretreatment (circles), or pretreated 30 minutes with 1 µM ionomycin (triangles), 12 hours with glucose-depleted Ringer (squares) or 2 hours with hyperosmotic shock by addition of 550 mM sucrose (diamonds).</p

    Coexpression of B-RAF increased hERG current in <i>Xenopus</i> oocytes.

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    <p><b>A.</b> Original tracings recorded in <i>Xenopus</i> oocytes injected with water (a), with cRNA encoding hERG alone (b) or with cRNA encoding hERG together with wild-type B-RAF (c). The <i>Xenopus</i> oocytes were depolarized from −80 mV holding potential to different voltages followed by a 500 ms repolarization to −60 mV evoking outward tail currents. <b>B.</b> Arithmetic means ± SEM (n = 12–47, arbitrary units) of the normalized outward tail current following a depolarization to +70 mV, recorded in <i>Xenopus</i> oocytes injected with water (white bar), with cRNA encoding hERG alone (light grey bar), or with cRNA encoding both, hERG and wild-type B-RAF (black bar). ***(p<0.001) indicates statistically significant difference from <i>Xenopus</i> oocytes expressing hERG channels alone. <b>C.</b> Arithmetic means ± SEM (n = 12–47, nA) of the peak tail current as a function of voltage in <i>Xenopus</i> oocytes injected with water (black triangles), with cRNA encoding hERG alone (white circles) or with cRNA encoding hERG and wild-type B-RAF (black circles). <b>D.</b> Arithmetic means ± SEM (n = 22–47, arbitrary units) of the normalized peak tail current as a function of voltage in <i>Xenopus</i> oocytes injected with cRNA encoding hERG alone (white circles) or with cRNA encoding hERG together with wild-type B-RAF (black circles).</p

    Downregulation of endometrial mesenchymal marker SUSD2 causes cell senescence and cell death in endometrial carcinoma cells

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    <div><p>The cause of death among the majority of endometrial cancer patients involves migration of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids into neighbouring organs. It is, thereby, important to identify factors that mediate metastasis. Cell adhesion and migration are modified by the mesenchymal stem cell (MSC) marker Sushi domain containing 2 (SUSD2), a type I transmembrane protein that participates in the orchestration of cell adhesion and migration through interaction with its partner Galactosidase-binding soluble-1 (LGALS1). MSCs have emerged as attractive targets in cancer therapy. Human endometrial adenocarcinoma (Ishikawa) cells were treated with TGFβ (10 ng/ml) for 72h. <i>SUSD2</i>, <i>LGALS1</i> and <i>MKI67</i> transcript levels were quantified using qRT-PCR. The proportion of SUSD2 positive (SUSD2+) cells and SMAD2/3 abundance were quantified by FACS and Western blotting, respectively. Senescent cells were identified with β-galactosidase staining; cell cycle and cell death were quantified using Propidium Iodide staining. Treatment of endometrial cancer cells (Ishikawa cells) with TGFβ (10 ng/ml) significantly decreased <i>SUSD2</i> transcript levels and the proportion of SUSD2 positive cells. Silencing of <i>SUSD2</i> using siRNA resulted in senescence and cell death of Ishikawa cells <i>via</i> activation of SMAD2/3. These findings suggest that SUSD2 counteracts senescence and cell death and is thus a potential chemotherapeutic target in human endometrial cancer.</p></div

    Coexpression of USP18 increases electrogenic peptide transport in PEPT2-expressing <i>Xenopus laevis</i> oocytes.

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    <p><b>A:</b> Representative original tracings showing I<sub>gly-gly</sub> in <i>Xenopus laevis</i> oocytes injected with water (a) or expressing USP18 alone (b) or expressing PEPT2 without (c) or with additional coexpression of wild type USP18 (d). <b>B:</b> Arithmetic means ± SEM (n = 8–9) of I<sub>gly-gly</sub> in <i>Xenopus</i> oocytes injected with water (striped bar), expressing USP18 alone (grey bar), or expressing PEPT2 without (white bar) or with (black bar) wild type USP18. **(p<0.01) indicates statistically significant difference from the absence of USP18.</p
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