Gain-of-Function p53N236S Mutation Drives the Bypassing of HRasV12-Induced Cellular Senescence via PGC–1α
Abstract
:1. Introduction
2. Results
2.1. The p53S/+ MEFs Escape HRasV12-Induced Senescence and Develop LOH
2.2. p53S Upregulates PGC–1α and Its Nuclear Translocation in Late-Stage p53S/++Ras Cells
2.3. p53S Improves Mitochondrial Quality and Quantity in Late-Stage p53S/++Ras Cells via PGC–1α
2.4. PGC–1α Downregulates Autophagy Levels in Late-Stage Cells by Reducing ROS Levels
2.5. p53S Regulates the Interaction between PGC–1α and PPARγ
3. Discussion
4. Materials and Methods
4.1. Mice and Tumor Cells Harvest
4.2. Cell Lines and Constructs
4.3. Western Blot Analysis
4.4. SA-β-Gal Staining
4.5. Oil Red O Staining
4.6. Disruption of the PGC–1α Gene through CRISPR/Cas9
- PCR primers #1: 5′-TTTCTTGCTTTCCCTTTTTCTG-3′ and 5′-ACCCCTATCCTCCCCACTAATA-3′,
- PCR primers #2: 5′-TTGATGCACTGACAGATGGAG-3′ and 5′-ACAGAATGGGCAAATCTAGGAA-3′,
- PCR primers #3: 5′-TCAACCCACTCATGTCTTCTGT-3′ and 5′-TACTAGAGACGGCTCTTCTGCC-3′,
- PCR primers #4: 5′-CCAGATCTTCCTGAACTTGACC-3′ and 5′-CTCCCCATACATCAGTCAGACA-3′.
4.7. Immunofluorescence and Microscopy Analysis
4.8. Real-Time PCR
- PGC–1α forward: 5′-AGCCGTGACCACTGACAACGAG-3′, reverse: 5′-GCTGCATGGTTCTGAGTGCTAAG-3′;
- Sod1 forward: 5′-CAAGCGGTGAACCAGTTGTG-3′, reverse: 5′-TGAGGTCCTGCACTGGTAC-3′;
- Sod2 forward: 5′-GCCTGCACTGAAGTTCAATG-3′, reverse: 5′-ATCTGTAAGCGACCTTGCTC-3′;
- Ucp2 forward: 5′-CAGGTCACTGTGCCCTTACCAT, reverse: 5′-CACTACGTTCCAGGATCCCAAG-3′;
- Ant forward: 5′-TTCCTGGCAGGTGGCATCG-3′, reverse: 5′-GGATTCTCACGACACAATCAATG-3′; and
- Catalase forward: 5′-ACCCTCTTATACCAGTTGGC-3′, reverse: 5′-GCATGCACATGGGGCCATCA-3′.
4.9. Chromatin Immunoprecipitation
4.10. ROS Detection
4.11. JC-10
4.12. ATP Detection Assay
4.13. MitoTrackerTM Green FM Staining
4.14. LysoTrackerTM Red DND-99 Staining
4.15. Co-IP Experiments
4.16. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
ALT | Alternative lengthening of telomere |
ChIP-on-Chip | Immunoprecipitation on chip |
DDR | DNA damage response |
DN | Dominant negative |
ER | Endoplasmic reticulum |
ES cells | Early-stage p53S/++Ras cells, senescent cells |
GOF | Gain of function |
IF | Immunofluorescence staining |
LFL | Li–Fraumeni like |
LFS | Li–Fraumeni syndrome |
LOF | Loss of function |
LOH | Loss of heterozygosity |
LS cells | Late-stage p53S/++Ras cells, which bypassed the OIS |
MEFs | Mouse embryonic fibroblasts |
OIS | Oncogene-induced senescence |
p53S | p53N236S |
PDAC | Pancreatic ductal adenocarcinoma |
PDH | Pyruvate dehydrogenase |
PGC–1α | PPARγ coactivator |
PPARγ | Peroxisome proliferator-activated receptor γ |
ROS | Reactive oxygen species |
SCID | Severe combined immune deficiency |
WT | Wild type |
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Yang, H.; Zhang, K.; Guo, Y.; Guo, X.; Hou, K.; Hou, J.; Luo, Y.; Liu, J.; Jia, S. Gain-of-Function p53N236S Mutation Drives the Bypassing of HRasV12-Induced Cellular Senescence via PGC–1α. Int. J. Mol. Sci. 2023, 24, 3790. https://doi.org/10.3390/ijms24043790
Yang H, Zhang K, Guo Y, Guo X, Hou K, Hou J, Luo Y, Liu J, Jia S. Gain-of-Function p53N236S Mutation Drives the Bypassing of HRasV12-Induced Cellular Senescence via PGC–1α. International Journal of Molecular Sciences. 2023; 24(4):3790. https://doi.org/10.3390/ijms24043790
Chicago/Turabian StyleYang, Hao, Ke Zhang, Yusheng Guo, Xin Guo, Kailong Hou, Jing Hou, Ying Luo, Jing Liu, and Shuting Jia. 2023. "Gain-of-Function p53N236S Mutation Drives the Bypassing of HRasV12-Induced Cellular Senescence via PGC–1α" International Journal of Molecular Sciences 24, no. 4: 3790. https://doi.org/10.3390/ijms24043790