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United States Department of Agriculture

Agricultural Research Service

Title: Proteins Present in Conditioned Media Are Altered in Dexamethasone-Inuced Adipogenesis of Primary Porcine Stromal-Vascular Cell Cultures.

Authors
item Poulos, Sylvia
item Hausman, Gary

Submitted to: Biomedical and Health Sciences Institute Spring Symposium
Publication Type: Abstract Only
Publication Acceptance Date: March 17, 2003
Publication Date: May 14, 2003
Citation: Poulos, S.P., Hausman, G.J. Proteins present in conditioned media are altered in dexamethasone-induced adipogenesis of primary porcine stromal-vascular cell cultures. Biomedical and Health Sciences Institute Spring symposium. 2003. p. 45.

Technical Abstract: The aim of this study was to identify proteins whose secretion is altered when adipogenesis is induced in S-V cells from postnatal pigs. S-V cells were isolated from subcutaneous adipose tissue from 5-7 day old postnatal pigs via collagenase digestion (n=4). Approximately 8x105 cells were plated on 100mm culture dishes with 16mL proliferating media. Upon confluency, media was aspirated, cells were rinsed with Dulbecco's Modified Eagle's Medium-F12 (DMEM), and differentiating media was added to each dish. Minimal adipogenesis was achieved using DMEM supplemented with 10% fetal bovine serum during the proliferative phase and DMEM supplemented with insulin, transferrin, and sodium selenite during the differentiation phase (FBS-ITS). Intermediate adipogenesis was achieved by treating cells with FBS-ITS supplemented with 80nM dexamethasone during the proliferative phase (FBS+Dex-ITS). Lastly, maximal adipogenesis was achieved by treating cells with FBS-ITS supplemented with 20nM dexamethasone during the differentiation phase (FBS-ITS+Dex). After three days, culture media was collected and frozen at 0°C. Media was concentrated using centrifugal filters with a 10kDa molecular weight cut off (Millipore). Two-dimensional differential in gel electrophoresis (2D-DIGE) of 40 µg protein from each sample was followed by image analysis using Decyder 4.0 software (Amersham Biosciences). Protein 'spots' with 2.5 fold difference in expression, as compared to FBS-ITS+Dex) were excised and mass spectroscopy was used to obtain tentative protein identification. This was followed by ToF/ToF mass spectroscopy for more definitive identification. The presence of 86 protein 'spots' were at least 2.5 fold different in media from minimal- or intermediate- adipogenic S-V cells when compared to maximally induced S-V cells (p<0.05). Of these, albumin, transferrin, and fetuin have been indentified using ToF/ToF mass spectroscopy. Discussion: The secretion of albumin, transferrin, and fetuin is increased with induction of adipogenesis. Large amounts of albumin present in conditioned media interfere with the identification of other proteins. The characterization of unknown proteins is, in part, limited to protein sequence information in available databases.

Last Modified: 10/26/2014
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