Abstract
Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA–protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.
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Abbreviations
- AEBSF:
-
4-(2-Aminoethyl)benzenesulfonyl fluoride
- anhCEC:
-
Adult normal human corneal epithelial cells
- anhK:
-
Adult normal human keratinocytes
- APS:
-
Ammonium persulfate
- ATP:
-
Adenosine triphosphate
- BCA:
-
Bicinchoninic acid
- ccDME-Ham:
-
Complete corneal epithelial cell culture medium
- ckDME-Ham:
-
Complete keratinocyte culture medium
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DMSO:
-
Dimethyl sulfoxide
- DNA:
-
Deoxyribonucleic acid
- DTT:
-
Dithiothreitol
- ECL:
-
Enhanced chemiluminescence
- EDTA:
-
Ethylenediaminetetraacetic acid
- EGF:
-
Epidermal growth factor
- EMSA:
-
Electrophoretic mobility shift assay
- Ham:
-
Ham’s medium
- HEPES:
-
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
- i3T3:
-
Irradiated Swiss 3T3
- iHFL:
-
Irradiated human feed layer
- MMP9:
-
Matrix metallopeptidase 9
- NE:
-
Nuclear extract
- NFI:
-
Nuclear factor I
- nhCEC:
-
Normal human corneal epithelial cell
- nhK:
-
Normal human keratinocytes
- nnhCEC:
-
Newborn normal human corneal epithelial cells
- nnhK:
-
Newborn normal human keratinocytes
- PAGE:
-
Polyacrylamide gel electrophoresis
- PBS:
-
Phosphate buffered saline
- PMSF:
-
Phenylmethylsulfonyl fluoride
- PNK:
-
T4 polynucleotide kinase
- poly(dI:dC):
-
Poly(deoxyinosinic-deoxycytidylic) acid sodium salt
- PVDF:
-
Polyvinylidene fluoride
- SCC:
-
Supershifted complexes
- SDS:
-
Sodium dodecyl sulfate
- Sp1:
-
Specificity protein 1
- STE:
-
Sodium Tris–HCl EDTA
- TBS:
-
Tris Buffered Saline
- TBS-T:
-
Tris Buffered Saline tween
- tDMEM:
-
Tissue transport medium
- TE:
-
Tris–HCl EDTA
- TEMED:
-
Tetramethylethylenediamine
- TM:
-
Melting temperature
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Acknowledgments
The authors would like to thank current and former members of the LOEX and CUO-Recherche laboratories who contributed to develop and improve the foregoing protocols.
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Le-Bel, G., Cortez Ghio, S., Larouche, D., Germain, L., Guérin, S.L. (2018). Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction. In: Turksen, K. (eds) Skin Stem Cells. Methods in Molecular Biology, vol 1879. Humana Press, New York, NY. https://doi.org/10.1007/7651_2018_153
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DOI: https://doi.org/10.1007/7651_2018_153
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