Identification of ETFB as a candidate protein that participates in the mechanoregulation of fibroblast cell number in collagen gel culture
| Autor: | Yasutomo Nishimori, Makoto Shimosaka, Shigenari Hirokawa, Hiroyuki Kitajima, Tomomasa Shimanuki |
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| Rok vydání: | 2011 |
| Předmět: |
Electron-Transferring Flavoproteins
Cell Count Dermatology Biology Biochemistry Extracellular matrix Fibrosis medicine Humans RNA Small Interfering Fibroblast Molecular Biology Cells Cultured Gel electrophoresis Differential display Two-dimensional gel electrophoresis Transfection Fibroblasts medicine.disease Molecular biology medicine.anatomical_structure Galectin-3 Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Collagen Stress Mechanical Gels |
| Zdroj: | Journal of dermatological science. 64(2) |
| ISSN: | 1873-569X |
| Popis: | Background Fibroblast activation is strongly influenced by mechanical environment in the wound-healing process, especially in fibrosis. Mechanically stressed three-dimensional collagen embedded culture is a useful model representing fibroblasts in morphological as well as biochemical situations encountered during fibrosis. Objective To find key proteins involved in reducing the number of fibroblasts during mechanical stress, we performed two-dimensional gel electrophoresis (2DE)-based differential display and siRNA-based functional screening with collagen gel culture focusing on the differences between attached and detached culture environments. Methods Membrane extracts of fibroblasts from 1 day of attached or detached cultures were subjected to 2DE. We compared protein expression levels and identified the attached-culture-dominant proteins by MALDI-TOF-MS. Next, fibroblasts were transfected with siRNA and embedded in collagen gel. Cell number was counted after 3 days in culture. Results Eight attached culture dominant proteins were identified with MALDI-TOF-MS. Transfection of siRNA against these proteins demonstrated that electron transfer flavoprotein β subunit (ETFB)-specific siRNA reduced the cell number in the attached culture without a decrease in the detached culture. Conclusion ETFB participates in the mechanoregulation of fibroblast cell number in collagen gel culture. |
| Databáze: | OpenAIRE |
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