Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC
Abstract
:1. Introduction
2. Results
2.1. UVC-Induced Cytotoxicity Is Similar in Mock and APP751-Expressing Cells at Low Doses
2.2. Effect of Aβ, H2O2 Treatment, and UVC Radiation on the NER-Associated Gene Expression in Mock and APP751-Expressing Cells
2.2.1. Basal NER-Associated Gene Expression in the Mock and APP751-Expression Cells
2.2.2. Effect of H2O2 Treatment on NER-Associated Gene Expression in Mock and APP751 Cell Lines
2.2.3. Effect of UVC Radiation on the NER-Associated Gene Expression in Mock and APP751-Expressing Cells
2.3. H2O2 Treatment Specifically Enhances Excision Capacity of UVC Lesions by the APP751 Cell Line
2.4. UVC Irradiation Induces a Similar Increase of Excision Capacity of UVC Lesions in Both Mock and APP751 Cell Lines
2.5. Assessments of the Photoproducts Repair Kinetics in the Mock and APP751-Expressing Cells with or without H2O2 Pre-Treatment
3. Discussion
4. Experimental Section
4.1. Cell Lines and Culture Conditions
4.2. UVC-Induced Cytotoxicity Measurements
4.3. Long-Term Toxicity of UVC
4.4. Preparation of Frozen Pellets
4.5. DNA Extraction and Digestion
4.6. Analysis of DNA Lesions (HPLC-MS/MS)
4.7. Gene Expression Analysis
4.7.1. mRNA Extraction and Quality Analysis
4.7.2. Reverse Transcription
4.7.3. Real Time Quantitative PCR
Name | GeneBank Entry | Forward Primer Sequence | Tm (°C) | Reverse Primer Sequence | Tm (°C) | Product Size (bp) |
---|---|---|---|---|---|---|
XPA | NM_000380.3 | gcagccccaaagataattga | 60.04 | tggcaaatcaaagtggttca | 60.09 | 183 |
XPC | NM_004628.4 | ccatgaggacacacacaagg | 60 | tccaatgaaccacttcacca | 59.94 | 187 |
DDB1 | NM_001923.3 | aacagagtggcgagagcatt | 60.02 | tcaatgacatgcagctcctc | 59.95 | 223 |
DDB2 | NM_000107.2 | tcaaggacaaacccaccttc | 59.94 | gtgaccaccattcggctact | 60 | 226 |
XPD | NM_000400.3 | gctggacatctaccccaaga | 60.07 | ccggatcacagcaatatcct | 59.92 | 166 |
XPB | NM_000122.1 | gcggcagagattcttggtag | 59.98 | ggccccagacatagaactca | 60.07 | 235 |
ERCC1 | NM_202001.1 | ttgtccaggtggatgtgaaa | 59.94 | gctggtttctgctcataggc | 59.98 | 151 |
PCNA | NM_002592.2 | ggctctagcctgacaaatgc | 59.98 | gcctccaacaccttcttgag | 59.84 | 224 |
4.7.4. Validation of Stably Expressed Housekeeping Genes
4.7.5. mRNA Quantification and Statistical Analyses
4.8. DNA Repair Assays
4.9. Cell Nuclear Extracts
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Forestier, A.; Douki, T.; De Rosa, V.; Béal, D.; Rachidi, W. Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC. Int. J. Mol. Sci. 2015, 16, 17422-17444. https://doi.org/10.3390/ijms160817422
Forestier A, Douki T, De Rosa V, Béal D, Rachidi W. Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC. International Journal of Molecular Sciences. 2015; 16(8):17422-17444. https://doi.org/10.3390/ijms160817422
Chicago/Turabian StyleForestier, Anne, Thierry Douki, Viviana De Rosa, David Béal, and Walid Rachidi. 2015. "Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC" International Journal of Molecular Sciences 16, no. 8: 17422-17444. https://doi.org/10.3390/ijms160817422