Journal: BMC Cancer

Article Title: Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin

doi: 10.1186/s12885-019-6219-7

Figure Lengend Snippet: Endothelial cell monolayer permeability assays using FITC-dextran. a ) Relative permeability 4 h after irradiation, compared to non-irradiated controls (0 Gy). b ) Relative permeability of cell monolayers measured 24 h after irradiation with 4 Gy, after treatment with VEGF-A (100 ng/ml) or TNFα (100 ng/ml) for 24 h, and after exposure to APMA (10 ng/ml) for 2 h, compared to vehicle (DMSO, 0.1%) only-treated controls. c ) Effects of ADAM inhibitors GI254023X (10 μM; specific for ADAM10 only) and GW280264X (10 μM; inhibits both ADAM10 and ADAM17). Inhibitor or vehicle were added to the monolayers 24 h before measurement. d) ADAM inhibitors counteract the irradiation-induced increase in permeability. Measurements were performed 24 h after addition of inhibitors and 4 h (left) or 24 h (right) after irradiation, respectively. Data shown are means ( n ≥ 3) and standard deviations. Statistics: t-test, ** p < 0.01, *** p < 0.001

Article Snippet: For Western blotting, primary antibodies reactive with the following antigens were used: P-β-catenin (Tyr142; diluted 1:500; #ab27798, abcam, Cambridge, UK); P-VEGF-R2 (Tyr1214; 1:1000, #AF1766, R&D Systems, Wiesbaden, Germany); VE-cadherin (BV9; 1:500; #sc-52,751, Santa Cruz Biotechnology, Heidelberg, Germany); VE-cadherin (1:1000; #2158S); ADAM10 (1:500–1:1000; #14194S); ADAM17 (1:1000; #3976S), β-catenin (1:1000; #9587S); VEGF-R2 (1:1000; #9698S); P-VEGF-R2 (Tyr1175; 1:1000; #2478S, all from Cell Signaling Technology, Frankfurt, Germany); and β-actin-POD (1:25,000; #A3854, Sigma-Aldrich).

Techniques: Permeability, Irradiation

Journal: BMC Cancer

Article Title: Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin

doi: 10.1186/s12885-019-6219-7

Figure Lengend Snippet: Irradiation-induced dislocalization and degradation of VE-cadherin and VEGF-A-induced activation of ADAM10. a – d ) Immunofluorescence stainings showing subcellular distribution of VE-cadherin in endothelial cells grown on coverslips. Upon reaching confluence, cells were mock-irradiated ( a ), irradiated with 4 Gy ( b and C ), or treated with 100 ng/ml VEGF-A ( d ) and prepared for VE-cadherin (green; Hoechst-33,342 nuclear staining is shown in blue) immunofluorescence microscopy after 2 h (B and D) or 24 h (C; 4 Gy only). Arrowheads indicate weakened or absent VE-cadherin staining at cell-cell contact sites. Asterisks mark areas of granular VE-cadherin staining indicating dislocation from cell-cell contact sites. E–H ) VE-cadherin localization in control and 4 Gy-irradiated endothelial cell layers in the absence or presence of the ADAM10-inhibitor GI254023X (10 μM). Cells were fixed and stained for VE-cadherin (green; nuclei are blue) after 24 h. Scale bars in A–H, 20 μm. I ) ADAM10 expression (precursor and mature form) in endothelial cells treated with irradiation (4 Gy; proteins isolated after 24 h) or VEGF-A (100 ng/ml; proteins isolated after 4 h) in the absence or presence of GI253023X (10 μM; added 30 min before treatments). Data (n ≥ 3) are shown as means ± standard deviations. Statistics: t-test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Western blotting, primary antibodies reactive with the following antigens were used: P-β-catenin (Tyr142; diluted 1:500; #ab27798, abcam, Cambridge, UK); P-VEGF-R2 (Tyr1214; 1:1000, #AF1766, R&D Systems, Wiesbaden, Germany); VE-cadherin (BV9; 1:500; #sc-52,751, Santa Cruz Biotechnology, Heidelberg, Germany); VE-cadherin (1:1000; #2158S); ADAM10 (1:500–1:1000; #14194S); ADAM17 (1:1000; #3976S), β-catenin (1:1000; #9587S); VEGF-R2 (1:1000; #9698S); P-VEGF-R2 (Tyr1175; 1:1000; #2478S, all from Cell Signaling Technology, Frankfurt, Germany); and β-actin-POD (1:25,000; #A3854, Sigma-Aldrich).

Techniques: Irradiation, Activation Assay, Immunofluorescence, Staining, Microscopy, Control, Expressing, Isolation

Journal: BMC Cancer

Article Title: Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin

doi: 10.1186/s12885-019-6219-7

Figure Lengend Snippet: MDA-MB-231 transendothelial migration and VEGF-A production. a ) Transendothelial cell migration assay showing the effect of endothelial cell irradiation (4 Gy) in the absence or presence of ADAM10/17 inhibitors on the transmigration of MDA-MB-231 breast tumor cells (n ≥ 3). b ) VEGF-A content in MDA-MB-231 cell culture supernatants measured by ELISA 24 h after irradiation (mock or 4 Gy; n ≥ 3). c and d ) Immunoblot analysis of VE-cadherin expression after irradiation (4 Gy), after treatment with recombinant VEGF-A (100 ng/ml), and after treatment with conditioned medium (CM; harvested after 24 h) from non-irradiated or irradiated (4 Gy) MDA-MB-231 cells (C; n = 2) and U-373 MG cells (D; n = 3) (lysates prepared after 24 h or 2 h in case of VEGF-A treatment). Data are absolute values ( b ) or relative to those of controls ( a , c ) and shown as means ± standard deviations. Statistics: t-test, * p < 0.05, ** p < 0.01

Article Snippet: For Western blotting, primary antibodies reactive with the following antigens were used: P-β-catenin (Tyr142; diluted 1:500; #ab27798, abcam, Cambridge, UK); P-VEGF-R2 (Tyr1214; 1:1000, #AF1766, R&D Systems, Wiesbaden, Germany); VE-cadherin (BV9; 1:500; #sc-52,751, Santa Cruz Biotechnology, Heidelberg, Germany); VE-cadherin (1:1000; #2158S); ADAM10 (1:500–1:1000; #14194S); ADAM17 (1:1000; #3976S), β-catenin (1:1000; #9587S); VEGF-R2 (1:1000; #9698S); P-VEGF-R2 (Tyr1175; 1:1000; #2478S, all from Cell Signaling Technology, Frankfurt, Germany); and β-actin-POD (1:25,000; #A3854, Sigma-Aldrich).

Techniques: Migration, Cell Migration Assay, Irradiation, Transmigration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Recombinant

Journal: Cancer Research

Article Title: Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

doi: 10.1158/0008-5472.can-08-3797

Figure Lengend Snippet: Figure 5. Cytoskeleton and VEGF-dependent signaling in cultured endothelial cells isolated from Shb knockout or wild-type liver. A, the cytoskeleton of isolated endothelial cells maintained in tissue culture for 5 to 7 d was visualized by staining with rhodamine-phalloidin, showing a less regular shape with numerous extensions in Shb null cells. B, corresponding wild-type control. No clear cytoskeletal difference between wild-type or knockout cells was noted after stimulation with VEGF-A (C, Shb knockout; D, Shb wild type). Addition of VEGF to the control cells (D) produced changes that made the cells resemble the Shb null cells in the absence of VEGF-A. Horizontal scale bar is shown. Equal amounts of protein from the experimental groups (+/, 20 ng/mL VEGF-A for 2 min) were subjected to Western blot analysis for the phosphorylated proteins indicated. Shb blot shows unstimulated cells only. The blots were subjected to densitometric analysis for phosphorylated FAK, total FAK, phosphorylated p38, total p38, pMLC, phosphorylated ERK, and total ERK in three separate experiments. Quantitation of the relative phosphorylation of FAK, MLC, p38, and ERK is also given. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01 (paired Students’ t test).

Article Snippet: The samples were electrophoresed on SDS-polyacrylamide gels, protein was transferred on to Hybond-P filters (GE Healthcare), and these were then probed for pY-1175 VEGFR-2, total VEGFR-2, pY-397 FAK, total FAK, phosphorylated extracellular signal-regulated kinase (ERK), total ERK, and phosphorylated Akt, total Akt, phosphorylated myosin light chain (pMLC), pY-658 VE-cadherin, phosphorylated p38, and total p38 (all antibodies were from Cell Signaling, except for total VEGFR-2 from R&D Systems, pY-397 FAK from Biosource, pY-VE-cadherin from ProSci, and pMLC from Santa Cruz Biotechnology) before incubation with secondary antibodies and enhanced chemiluminescence.

Techniques: Cell Culture, Isolation, Knock-Out, Staining, Control, Produced, Western Blot, Quantitation Assay, Phospho-proteomics

Journal: The Journal of international medical research

Article Title: Immunohistochemical levels of cyclo-oxygenase-2, matrix metalloproteinase-9 and vascular endothelial growth factor in papillary thyroid carcinoma and their clinicopathological correlations.

doi: 10.1177/0300060513505485

Figure Lengend Snippet: Figure 1. Representative photomicrographs showing immunohistochemical staining for cyclo-oxygenase-2, matrix metalloproteinase-9 and vascular endothelial growth factor proteins in benign thyroid adenomas (A, C and E, respectively) and in papillary thyroid carcinomas (B, D and F, respectively). The colour version of this figure is available at: http://imr.sagepub.com.

Article Snippet: After blocking with 3% bovine serum albumin (Wuhan Boster Biological Technology) in 0.01mM phosphate buffered saline (PBS; pH 7.2) for 2 h at 37 C, the sections were incubated with mouse monoclonal antibodies against human COX-2,MMP-9 and VEGF proteins (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500 overnight at 4 C. The samples were then washed three times in 0.01mM PBS (pH 7.2) for 5min.

Techniques: Immunohistochemical staining, Staining

Journal: Science Advances

Article Title: Ultraefficient extracellular vesicle–guided direct reprogramming of fibroblasts into functional cardiomyocytes

doi: 10.1126/sciadv.abj6621

Figure Lengend Snippet: ( A ) Time-course analysis of cardiac precursor marker expression by RT-qPCR. “H” represents mRNA from the heart at embryonic day 13.5 (positive control) ( n = 3 separate experiments). ( B ) Immunostaining for the markers Isl1 and Ki67 in single cells digested from clusters at day 15 after induction. ( C and D ) Continuous culture of Isl1 + cells in either smooth muscle cell differentiation medium or endothelial cell differentiation medium for an additional 2 weeks produced Cnn2 + /α-SMA + cells (C) and VE-cadherin + /PECAM + cells (D), respectively. ( E and F ) Quantitative RT-PCR analysis of the indicated markers in induced smooth muscle cells (iSMs) (E) or induced endothelial cells (iEndos) ( n = 3 separate experiments) (F). ( G ) Phase-contrast (top) and fluorescence microscopy (bottom) time-lapse images of alkaline phosphatase (AP) live-stained iCMs generated from MEFs. iCMs were immunostained for cTnT (red) on day 28. ( H ) AP live staining of undifferentiated ESCs (positive control). ( I ) RT-qPCR analysis showing the time course of the expression of the pluripotency markers Nanog and Rex1 ( n = 3 separate experiments). All data are presented as means ± SD (*** P < 0.001, ** P < 0.01, and * P < 0.05, two-sided t test). Scale bars, 100 μm (B to D), 300 μm (G), and 200 μm (H).

Article Snippet: Subsequently, the cells were blocked with 5% BSA for 1 hour at room temperature and stained with 1:200 mouse immunoglobulin G (IgG) α-MHC (ab50967), 1:200 mouse IgG cTnT (ab8295), 1:100 rabbit IgG cTnI (ab47003), 1:200 rabbit IgG GATA4 (ab84593), 1:200 rabbit IgG Mef2c (ab64644), 1:200 mouse IgG Nkx-2.5 (ab91196), 1:200 rabbit IgG Connexin-43 (ab11370), 1:100 polyclonal rabbit IgG MLC2v (ab79935), 1:200 monoclonal mouse IgG Ki67 (ab8191, all Abcam), 1:100 monoclonal rabbit IgG α-actinin (7H1L69, Thermo Fisher Scientific), 1:300 monoclonal mouse IgG MLC2a (#311011, Synaptic systems), 1:100 polyclonal rabbit IgG Isl1 (LS-C334676, LifeSpan BioSciences), 1:400 monoclonal mouse IgG SMA (A2547, Sigma-Aldrich), 1:100 polyclonal goat IgG Calponin 2 (sc-16607), 1:100 polyclonal goat IgG PECAM (sc-1506), or 1:100 monoclonal mouse IgG VE-cadherin (sc-9989, all Santa Cruz Biotechnology) overnight at 4°C in 1% BSA in PBS.

Techniques: Marker, Expressing, Quantitative RT-PCR, Positive Control, Immunostaining, Cell Differentiation, Produced, Fluorescence, Microscopy, Staining, Generated