Crosstalk between BER and NHEJ in XRCC4-Deficient Cells Depending on hTERT Overexpression
Abstract
:1. Introduction
2. Results
2.1. XRCC4 Knockdown Caused NHEJ Deficiency in Normal TIG-1 Cells
2.2. Effect of XRCC4 Knockdown on Expression of BER Components in Normal TIG-1 Cells
2.3. Effects of XRCC4 Knockdown on BER and NHEJ Expression in hTERT Modified NBE1 Cells
2.4. Analysis of DNA Damage after XRCC4 Knockdown in Normal and hTERT-Modified Cells
2.5. Effect of XRCC4 Knockdown on PARP1 Expression in Normal and hTERT-Modified Cells
2.6. The Effect of XRCC4 Knockdown on Transcription Factors p53 and Sp1 in Normal and hTERT Modified Cells
2.7. Cell Cycle Alterations after XRCC4 Knockdown in Normal and hTERT-Modified Cells
3. Discussion
4. Materials and Methods
4.1. Cells and Cell Culture
4.2. siRNA Transfections
4.3. Real-Time PCR (qPCR)
4.4. Western Blot
4.5. Alkaline Comet Assay
4.6. Image Analysis
4.7. Cell Cycle Analysis
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Target | Sequence (5′ to 3′) |
---|---|
XRCC4 | AAUCUUGGGACAGAACCUAAA |
Target | Forward Primer (5′→3′) | Reverse Primer (5′→3′) |
---|---|---|
B2M | ATGTCTCGCTCCGTGGCCTTA | ATCTTGGGCTGTGACAAAGTC |
XRCC1 | CACGGATGAGAACACGGACA | TCCCCATTGAAGGCTGTGAC |
LIG4 | GAAGGCATCTGGTAAGCTCG | TTTCTTCATCTTTGGGGCAG |
XRCC4 | TGCAAAGAAATCTTGGGACAG | TGCTCCTTTTTAGACGTCTC |
Ku70 | GTTGATGCCTCCAAGGCTATG | CCCCTTAAACTGGTCAAGCTCTA |
Ku80 | TGGAAGTGTGAATCCTGCTG | TCCAAAAACTGTTCGATGTGA |
Sp1 | GCCTCCAGACCATTAACCTCAG | TCATGTATTCCATCACCACCAG |
p53 | GACATAGTGTGGTGGTGCCC | CAAAGCTGTTCCGTCCCAGT |
APE1 | GGGCGTTCGTAACGGGAATG | TGCGGCCGTCTTACTCTTCT |
LIG3 | TGTGTTAGACGCCCTTGACC | TACTCAACGGACTTGCAGGC |
Target | Antibodies |
---|---|
XRCC1 | MS-1993-P0 (Thermo Scientific, Wilmington, DE, USA) |
DNA Ligase 3 | GTX70143 (GeneTex, Irvine, CA, USA) |
APE1 | NB100-101 (Novus Biologicals, Toronto, ON, Canada) |
XRCC4 | 66621-1-Ig (Proteintech, Solana Beach, CA, USA) |
DNA Ligase 4 | ab193353 (Abcam, Waltham, MA, USA) |
Ku80 | ab119935 Abcam (Abcam, Waltham, MA, USA) |
Ku70 | NB100-1915 (Novus Biologicals, Toronto, ON, Canada) |
Sp1 | 07-645 (Merck Millipore, Carrigtwohill, Ireland) |
p53 | sc-126 (SANTA CRUZ, Dallas, TX, USA) |
p21 Waf1/Cip1 (12D1) | #2947 (Cell signaling technology, Danvers, MA, USA) |
PARP1 | ab227244 Abcam (Abcam, Waltham, MA, USA) |
β-actin | ab6276 Abcam (Abcam, Waltham, MA, USA) |
α-tubulin | T6199 (Merck KGaA, Darmstadt, Germany) |
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Sergeeva, S.V.; Loshchenova, P.S.; Oshchepkov, D.Y.; Orishchenko, K.E. Crosstalk between BER and NHEJ in XRCC4-Deficient Cells Depending on hTERT Overexpression. Int. J. Mol. Sci. 2024, 25, 10405. https://doi.org/10.3390/ijms251910405
Sergeeva SV, Loshchenova PS, Oshchepkov DY, Orishchenko KE. Crosstalk between BER and NHEJ in XRCC4-Deficient Cells Depending on hTERT Overexpression. International Journal of Molecular Sciences. 2024; 25(19):10405. https://doi.org/10.3390/ijms251910405
Chicago/Turabian StyleSergeeva, Svetlana V., Polina S. Loshchenova, Dmitry Yu. Oshchepkov, and Konstantin E. Orishchenko. 2024. "Crosstalk between BER and NHEJ in XRCC4-Deficient Cells Depending on hTERT Overexpression" International Journal of Molecular Sciences 25, no. 19: 10405. https://doi.org/10.3390/ijms251910405