CDK5RAP3, a Novel Nucleoplasmic Shuttle, Deeply Regulates HSF1-Mediated Heat Stress Response and Protects Mammary Epithelial Cells from Heat Injury
Abstract
:1. Introduction
2. Results
2.1. CDK5RAP3 and HSPs Showed Similar Expression Trend in BMECs after Exposure to Heat Stress
2.2. The Deletion of the CDK5RAP3 Gene Accelerated the Death of Mice
2.3. CDK5RAP3 Induces Apoptosis Due to the Heat Stress
2.4. Regulation of the Heat Shock Factor 1-Mediated Stress Response Pathway by CDK5RAP3
2.4.1. CDK5RAP3 Depletion Inhibited the Expression of HSP90, While HSP70 Showed No Significant Change
2.4.2. The Knockdown of CDK5RAP3 Did Not Affect the mRNA Expression Level of HSF1, But the Expression of HSF1 Protein
2.4.3. The Knockout of CDK5RAP3 Gene Affected the Nuclear and Cytoplasmic Distribution of HSF1 and Inhibited the Trimer Formation of HSF1
2.4.4. CDK5RAP3 Interacts with HSF1 to Influence the Expression of HSP90
2.4.5. The Knockdown of HSF1 Affect the mRNA and Protein Expression Level of CDK5RAP3 in BMECs
2.4.6. Immunofluorescence Results Show that CDK5RAP3 Binds to HSF1
2.5. Alteration in Expression of GRP78 and CHOP Due to Deficiency of CDK5RAP3 and Heat Stress
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Chemical Reagents
4.3. Cell Culture and Heat Treatment
4.4. Identification of Bovine Mammary Epithelial Cells by Immunofluorescence
4.5. Total RNA Isolation and qRT-PCR Analysis
4.6. The Extraction of Cellular Nuclear and Cytoplasmic Protein
4.7. The Detection of Tissue and Cellular Protein Concentration
4.8. Western Blot Analysis
4.9. Trimerization Detection Experiment
4.10. Co-Immunoprecipitation
4.11. Immunofluorescent Staining
4.12. Statistical Method
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequences |
---|---|
mβ-ACTIN | Forward: 5′-GACCTCTATGCCAACACAGT-3′ |
Reverse: 5′-AGTACTTGCGCTCAGGAGGA-3′ | |
mGAPDH | Forward: 5′-AACTTTGGCATTGTGGAAGG-3′ |
Reverse: 5′-ACACATTGGGGGTAGGAACA-3′ | |
mCDK5RAP3 | Forward: 5′-ATGAGATCGACTGGGGTGAC-3′ |
Reverse: 5′-AGCCTCAGTTCCTGTCTCCA-3′ | |
mHSF1 | Forward: 5′-TCATCTGCTGGAGCCCGAGTG-3′ |
Reverse: 5′-ATGTTGAGCTGCCGCACGAAG-3′ | |
bβ-ACTIN | Forward: 5′-GGGCAGGTCATCACCATCGG-3′ |
Reverse: 5′-TCATTGTGCTGGGTGCCAGG-3′ | |
bGAPDH | Forward: 5′-AAGGTCGGAGTGAAC-3′ |
Reverse: 5′-CGTTCTCTGCCTTGACTGTG-3′ | |
bCDK5RAP3 | Forward: 5′-TCGACTGGCTGGTAGACAGAAGG-3′ |
Reverse: 5′-GTCCTGGATGGCGGCATTGATC-3′ | |
bHSP90 | Forward: 5′-CCAAGTCTGGCACTAAAG-3′ |
Reverse: 5′-GAAGACTCCCAAGCATAC-3′ | |
bHSF1 | Forward: 5′-AAAGATCCCCCTGATGCTGAACGAC-3′ |
Reverse: 5′-CAGTTCGGTGATGTCGGAGATGATG-3′ |
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Shen, Y.; Zou, Y.; Li, J.; Chen, F.; Li, H.; Cai, Y. CDK5RAP3, a Novel Nucleoplasmic Shuttle, Deeply Regulates HSF1-Mediated Heat Stress Response and Protects Mammary Epithelial Cells from Heat Injury. Int. J. Mol. Sci. 2020, 21, 8400. https://doi.org/10.3390/ijms21218400
Shen Y, Zou Y, Li J, Chen F, Li H, Cai Y. CDK5RAP3, a Novel Nucleoplasmic Shuttle, Deeply Regulates HSF1-Mediated Heat Stress Response and Protects Mammary Epithelial Cells from Heat Injury. International Journal of Molecular Sciences. 2020; 21(21):8400. https://doi.org/10.3390/ijms21218400
Chicago/Turabian StyleShen, Yangyang, Yan Zou, Jun Li, Fanghui Chen, Honglin Li, and Yafei Cai. 2020. "CDK5RAP3, a Novel Nucleoplasmic Shuttle, Deeply Regulates HSF1-Mediated Heat Stress Response and Protects Mammary Epithelial Cells from Heat Injury" International Journal of Molecular Sciences 21, no. 21: 8400. https://doi.org/10.3390/ijms21218400