Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Cell Culture and Compound Preparation
2.3. Cell Viability Assay
2.4. mRNA Expression Analysis by Semiquantitative Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR) and Quantitative Real-Time PCR (qPCR)
2.5. siRNA Transfection
2.6. Preparation of Whole-Cell Lysates, Nuclear Fraction, and Immunoblotting
2.7. Immunoprecipitation-Kinase Assay
2.8. Cellular Thermal Shift Assay (CETSA)
2.9. Statistical Analysis
3. Results
3.1. 8-HD Downregulates IRF-3-Dependent Genes in LPS-Stimulated RAW264.7 Cells
3.2. 8-HD Inhibits Phosphorylation IRF-3 and Nuclear Translocation of IRF-3
3.3. 8-HD Alters Upstream Signaling Enzymes of the IRF-3 Signaling Pathway
3.4. 8-HD Inhibits the Kinase Activity of IKKε to Phosphorylates IRF-3
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
8-HD | 8-hydroxydaidzein |
NF-κB | nuclear factor-κB |
AP-1 | activator protein-1 |
IRF-3 | interferon regulatory factor -3 |
LPS | lipopolysaccharide |
AKT | protein kinase B |
TRAF3 | TNF receptor associated factor 3 |
IFN-β | Interferon-β |
CXCL10 | (C-X-C motif) chemokine 10 |
PAMPs | pathogen-associated molecular patterns |
PRRs | pattern recognition receptors |
TLRs | Toll-like receptors |
RLRs | RIG-I-like receptors |
cGAS | cyclic GMP-AMP synthase |
STING | stimulators of interferon genes |
CCL5 | (C-C motif) ligand 5 |
Nrf2 | Nuclear factor erythroid 2–related factor 2 |
TAK1 | Transforming growth factor beta-activated kinase 1 |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
IKKε | inhibitor of nuclear factor kappa-B kinase subunit epsilon |
TBK1 | TANK binding kinase 1 |
ATP | Adenosine 5′-triphosphate |
TRIF | TIR-domain-containing adapter-inducing interferon-β |
TRAM | TRIF–related adaptor molecule |
MyD88 | Myeloid differentiation primary response 88 |
PI3K | Phosphatidylinositol-4,5-bisphosphate 3-kinase |
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PCR Type | Genes Name | Sequence (5′-3′) | |
---|---|---|---|
Semiquantitative RT-PCR | IFN-β | Forward | TCCAAGAAAGGACGAACATT |
Reverse | TGAGGACATCTCCCACGTCA | ||
IFIT1 | Forward | ATGCAGTCGTAGCCTATCGC | |
Reverse | CCTGCAAGGCCCTGTTTAGA | ||
GAPDH | Forward | ACCACAGTCCATGCCATCAC | |
Reverse | CCACCACCCTGTTGCTGTAG | ||
qPCR | IFN-β | Forward | AAGAGTTACACTGCCTTTGCTATC |
Reverse | CACTGTCTGCTGGTGGAGTTCATC | ||
IKKε | Forward | CCACTTGGAGTGCAGGAAGA | |
Reverse | CCGGAT TTCTTGTTTCGGGC | ||
CXCL10 | Forward | ATCATCCCTGCGAGCCTATCC | |
Reverse | CGGATTCAGACATCTCTGCTCATC | ||
IFIT1 | Forward | ATGCAGTCGTAGCCTATCGC | |
Reverse | CCTGCAAGGCCCTGTTTAGA | ||
GAPDH | Forward | CAATGAATACGGCTACAGCA | |
Reverse | AGGGAGATGCTCAGTGTTGG |
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Aziz, N.; Kang, Y.-G.; Kim, Y.-J.; Park, W.-S.; Jeong, D.; Lee, J.; Kim, D.; Cho, J.Y. Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages. Biomolecules 2020, 10, 238. https://doi.org/10.3390/biom10020238
Aziz N, Kang Y-G, Kim Y-J, Park W-S, Jeong D, Lee J, Kim D, Cho JY. Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages. Biomolecules. 2020; 10(2):238. https://doi.org/10.3390/biom10020238
Chicago/Turabian StyleAziz, Nur, Young-Gyu Kang, Yong-Jin Kim, Won-Seok Park, Deok Jeong, Jongsung Lee, Donghyun Kim, and Jae Youl Cho. 2020. "Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages" Biomolecules 10, no. 2: 238. https://doi.org/10.3390/biom10020238
APA StyleAziz, N., Kang, Y.-G., Kim, Y.-J., Park, W.-S., Jeong, D., Lee, J., Kim, D., & Cho, J. Y. (2020). Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages. Biomolecules, 10(2), 238. https://doi.org/10.3390/biom10020238