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Sign Up. All rights reserved. The email address is not valid. This field is required. Create an Account. In consequence, pro or anti-tumor activities of ADAMTS could also be influenced by factors other than interaction with fibulin For instance, these interactions may underlie the molecular mechanisms by which some tumor-protective metalloproteases exert their anti-tumor activities.
Additionally, tumor susceptibility assays using mice lacking both Adamts12 and Fbln2 genes would be necessary to understand in depth the significance of this interaction not only in breast cancer but also in tumors from different origins. The Matchmaker Two-hybrid System and a human fetal kidney library prey vectors containing a sequence enconding amino acids of the GAL4 protein AD were purchased from Clontech. Both bait and prey vectors were introduced into Saccharomyces cerevisiae AH cells and selection of double transformants was performed on synthetic minimal medium lacking leucine, tryptophan and histidine.
Identification of the prey inserts was performed by direct sequencing. Anti-actin antibody was from Sigma-Aldrich.
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Immunoreactive proteins were visualized using HRP-peroxidase labeled anti-rabbit or anti-mouse secondary antibody and the ECL detection system Pierce. Relative expression levels were quantified and normalized to actin levels within the same blot using the Image J software. MCF-7 cells extract producing fibulin-2 was used as a negative control. As indicated, different amounts of fibulin-2 were added to the reaction.
Peptide degradation was examined by western blot using an anti-His antibody Quiagen. For MCF-7 cells, 2. Cells that reached the lower surface were stained with crystal violet. At least three independent experiments were performed with triplicates for each condition. Cells were counted in eight randomly selected microscopic fields.
Briefly, 1,25 x 10 4 cells were seeded per well and migration was monitored by time-lapse microscopy using a Zeiss Axio Observer Microscopy. Experiments were performed with triplicates and covered area was quantified at different times using Image J. After 7 days, mammospheres were collected and enzymatically dissociated as previously described [ 43 ].
Mammospheres formation was monitored microscopically daily to ensure they derived from single cells and not from aggregates. Number of mammospheres was determined after 7 days culture. As a control, the opposite flank in each animal was injected with identical number of control MCF-7 cells transfected with an empty vector.
Tumor growth was monitored weekly as previously described [ 29 ]. For estrogen supplementation, pellets of 1. Mice were housed under specific pathogen-free conditions and following the guidelines of the Committee on Animal Experimentation of the Universidad de Oviedo, Asturias, Spain. Selected slides were counterstained with haematoxylin. The intensity of immunostaining was evaluated by two independent observers directly upon microscope and was scored as absent, slight, moderate and strong. Study methodologies were conformed to the standards set by the Declaration of Helsinki and were approved by the Ethics Committee of the Universidad de Oviedo-Principado de Asturias Spain.
To assess the effect of ADAMTS and fibulin-2 on breast cancer prognosis, survival probability was determined using the data available at www. Results were represented as Kaplan-Meier long-rank test survival plots.
Statistical analysis were carried out using the GraphPad Prism 5. The occurrence of significant differences was determined with the Student-Welch t test. We thank Dr.
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National Center for Biotechnology Information , U. Journal List Oncotarget v. Published online Jan Esteban , 7 Alvaro J. Obaya , 2, 7 and Santiago Cal 1, 2. Pedro M. Jose A. Manuel M. Alvaro J. Author information Article notes Copyright and License information Disclaimer. Correspondence to: Santiago Cal , se. Received Dec 12; Accepted Jan This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. Associated Data Supplementary Materials oncotargets Abstract Balance between pro-tumor and anti-tumor effects may be affected by molecular interactions within tumor microenvironment.
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Open in a separate window. Figure 1. Figure 2. Figures 3. Figure 4. Figure 5. Figure 6. Survival analysis To assess the effect of ADAMTS and fibulin-2 on breast cancer prognosis, survival probability was determined using the data available at www. Statistical analysis Statistical analysis were carried out using the GraphPad Prism 5.
Click here to view. Acknowledgments We thank Dr. The extracellular matrix: a dynamic niche in cancer progression. J Cell Biol.
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