Journal: Cancers

Article Title: Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover

doi: 10.3390/cancers13225673

Figure Lengend Snippet: Deregulation of MCAK impairs the cell spreading and adhesion capacity . ( A ) Schedule of cell spreading/adhesion assay. ( B , C ) RPE CRISPRi/a cells were stained for active integrin (red), paxillin (green), and DNA (DAPI, blue). Representatives of 20 min and 60 min cell re-adhesion are shown. Scale: 25 µm; inset scale: 10 µm. ( D , G ) Quantification of cell size of RPE CRISPRi/a sgcon and sgMCAK cells at 20 min ( D ) and 60 min ( G ) after reseeding. The results are based on three independent experiments and presented as scatter plots showing mean ± SEM (180 cells). ( E , H ) Quantification of the mean fluorescence intensity of paxillin after 20 min ( E ) and 60 min ( H ) after reseeding (180 FAs measured in each experiment). The results are based on three independent experiments and presented as scatter plots showing mean ± SEM. ( F , I ) Measurement of the paxillin signal size after the cells were reseeded for 20 min ( F ) and 60 min ( I ) (at least 60 FAs measured in each experiment). The results are based on three independent experiments and presented as scatter plots showing mean ± SEM. ( J , K ) Quantified percentage of fully re-attached cells after 20 min ( J ) and 60 min ( K ) (50 cells measured in each experiment). The results are based on three independent experiments and presented as box plots showing mean ± SEM. Unpaired Mann–Whitney U test for ( D – I ). Student’s t -test for ( J , K ). * p < 0.05, ** p < 0.01, and *** p < 0.001. Abbreviation: n.s.: not significant.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against pericentrin (#ab28144, Abcam, Cambridge, UK), human immune serum against human anti-centromere (#HCT-0100, ImmunoVision, Springdale, Germany), mouse monoclonal FITC-conjugated antibody against α-tubulin (#F2168, Sigma-Aldrich, Taufkirchen, Germany), rabbit polyclonal antibody against α-tubulin (#ab15246, Abcam, Cambridge, UK), rabbit polyclonal antibody against FAK (#3285, Cell Signaling, Frankfurt, Germany), mouse monoclonal antibody against p-FAK (Y397) (#8556, Cell Signaling, Frankfurt, Germany), monoclonal mouse antibody against paxillin (#610052, BD Biosciences, Heidelberg, Germany) and rabbit monoclonal antibody against p-paxillin (Y118) (#2541, BD Biosciences, Heidelberg, Germany) and rat monoclonal antibody against active β1-integrin (CD29) (#9EG7, BD Pharmingen™, San Jose, CA, USA).

Techniques: Cell Adhesion Assay, Staining, Fluorescence, MANN-WHITNEY

Journal: Experimental Physiology

Article Title: Effects of soluble guanylyl cyclase stimulation on muscle oxygenation and exercise capacity in heart failure with mildly reduced ejection fraction

doi: 10.1113/EP092756

Figure Lengend Snippet: Spinotrapezius muscle sGCβ1 (a) and SOD1 (b) protein expression. Data are means ± SD. Twenty‐one samples of spinotrapezius muscle (Healthy, n = 4; Healthy + BAY41, n = 4; HFmrEF, n = 6; HFmrEF + BAY41, n = 7) were used to measure sGCβ1 and SOD1 expression via western immunoblotting. Representative western blot images are presented below each graph. (a) There was a significantly greater expression of sGCβ1 in the spinotrapezius muscle of HFmrEF + BAY41 rats (* p = 0.045). (b) No statistical difference was detected in spinotrapezius muscle SOD1 expression (Healthy, p = 0.932; HFmrEF, p = 0.897). Data were analysed via two‐way mixed‐effects ANOVA and Fisher's least significant difference test without correction for multiple comparisons. Abbreviations: a.u., arbitrary units; BAY41, BAY 41‐2272; HFmrEF, heart failure with mildly reduced ejection fraction; sGCβ1, soluble guanylyl cyclase beta 1 subunit; SOD1, superoxide dismutase 1.

Article Snippet: Protein quantification in medial costal diaphragm lysates was completed with Jess Simple Western (ProteinSimple) automated western blot analysis with primary antibodies for soluble guanylyl cyclase subunit beta 1 (sGCβ1; NBP1‐89784, Novus Biologicals, 1:10 dilution) and superoxide dismutase 1 (SOD1; NBP1‐90186, Novus Biologicals, 1:100 dilution).

Techniques: Expressing, Western Blot

Journal:

Article Title: Physiological levels of tumstatin, a fragment of collagen IV ?3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via ?V?3 integrin

doi:

Figure Lengend Snippet: A: Proliferation assay in β3 integrin−/− endothelial cells. MLEC were isolated from wild-type (β3+/+) and β3 integrin−/− (β3−/−) mice as previously described (Maeshima et al., 2002). Proliferation was assessed by [3H]-thymidine incorporation. All groups represent triplicate analyses. The results are shown as the mean ± SEM. ** indicates p < 0.01; compared to control cells.

Article Snippet: They were incubated with various primary antibodies, i.e., rat anti-mouse CD31 (Pharmingen), rabbit anti-von Willebrand Factor (vWF) (Dako), rat anti-β1 integrin (Pharmingen), and hamster anti-β3 integrin (Pharmingen) antibodies at room temperature for 2 hr.

Techniques: Proliferation Assay, Isolation

Journal:

Article Title: Physiological levels of tumstatin, a fragment of collagen IV ?3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via ?V?3 integrin

doi:

Figure Lengend Snippet: A: Immunohistochemical staining with anti-β3 integrin (β3) antibody and CD31 or vWF was performed to assess localization of β3 integrin in blood vessels. Anti-CD31 antibody was used for the tumor and skin tissues and anti-vWF antibody was used for the liver tissue. β3 integrin-positive blood vessels (merge) are indicated with arrows. Scale bar: 50 μm.

Article Snippet: They were incubated with various primary antibodies, i.e., rat anti-mouse CD31 (Pharmingen), rabbit anti-von Willebrand Factor (vWF) (Dako), rat anti-β1 integrin (Pharmingen), and hamster anti-β3 integrin (Pharmingen) antibodies at room temperature for 2 hr.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Physiological levels of tumstatin, a fragment of collagen IV ?3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via ?V?3 integrin

doi:

Figure Lengend Snippet: A: Immunohistochemical staining with anti-β3 integrin (β3) antibody and CD31 was performed to assess localization of β3 integrin in the tumor blood vessels with volumes of 0.1, 0.5 and 3.0 cm3 in wild-type mice. β3 integrin-positive blood vessels (merge) are indicated with arrows. Scale bar: 50 μm.

Article Snippet: They were incubated with various primary antibodies, i.e., rat anti-mouse CD31 (Pharmingen), rabbit anti-von Willebrand Factor (vWF) (Dako), rat anti-β1 integrin (Pharmingen), and hamster anti-β3 integrin (Pharmingen) antibodies at room temperature for 2 hr.

Techniques: Immunohistochemical staining, Staining

Journal: Cell Host & Microbe

Article Title: β1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections

doi: 10.1016/j.chom.2017.05.001

Figure Lengend Snippet:

Article Snippet: The samples were stained with a rat anti-mouse β1-integrin (1:100 dilution, clone MB1.2, Merck Millipore), anti-acid ceramidase (1:100 dilution), anti-ceramide (1:100 dilution), anti-sphingosine (1:1000 dilution), anti-mouse β2-integrin antibodies (1:100, clone M1812, 1:100, #557437, BD) or FITC-Annexin (1:200, #11 828 681 001, Roche) in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl 2 , 0.7 mM MgCl 2 , 0.8 mM MgSO 4 ) plus 1% FCS at room temperature for 45 min.

Techniques: Virus, Isolation, Recombinant, Software