TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Antibodies
2.1.1. Chemicals
2.1.2. Antibodies
2.2. Cell Lines
2.3. Methods
2.3.1. Transfection
2.3.2. Generation of Stable Cells
2.3.3. Cell Proliferation Assay
2.3.4. Live/Dead Cell Staining Using Propidium Iodide (PI)
2.3.5. Brefeldin A Treatment
2.3.6. Immunofluorescence
2.3.7. Quantitation of Immunostaining
2.3.8. Counting of Ki67-Positive Cells and Plotting the Graphs
2.3.9. Colocalization Analysis
2.3.10. Phase Contrast Imaging
2.3.11. Western Blotting
2.3.12. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR
Component | Amount |
RNA | x µL |
10 mM dNTP | 1 µL |
Random hexamers (50 ng/µL) | 2 µL |
DEPC water | to make 10 µL |
Component | Amount |
10× RT buffer | 2 µL |
25 mM MgCl2 | 4 µL |
0.1 M DTT | 2 µL |
RNaseOUT™ (ribonuclease inhibitor) | 1 µL |
2.3.13. TaqMan RT-PCR
Component | Amount |
TaqMan™ Fast Advanced Master Mix (2×) | 5 µL |
TaqMan™ Assay (20×) | 0.5 µL |
Nuclease-free water | 2.5 µL |
cDNA | 2 µL |
2.3.14. Immunoprecipitation
2.4. Statistical Analyses
3. Results
3.1. TLR2 and TLR9 Intrabodies Cause Pancreatic Cancer Cell Death
3.2. TLR2 and TLR9 Intrabody-Mediated Pancreatic Cancer Cell Death through Inhibition of the STAT3 Phosphorylation Pathway
3.3. Inhibition of TLR2 and TLR9 by Its Specific Intrabodies Blocks Inflammatory Mediators IL-6 and TNF-α
3.4. TLR2 and TLR9 Intrabodies Bind to TLR2 and TLR9 Proteins to Retain These Proteins in the Endoplasmic Reticulum
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Name | Assay ID |
---|---|
IL-6 | Hs00174131_m1 |
TNFα1 | Hs00855471_g1 |
GAPDH | Hs99999905_m1 |
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Ajay, A.K.; Gasser, M.; Hsiao, L.-L.; Böldicke, T.; Waaga-Gasser, A.M. TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth. Antibodies 2024, 13, 11. https://doi.org/10.3390/antib13010011
Ajay AK, Gasser M, Hsiao L-L, Böldicke T, Waaga-Gasser AM. TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth. Antibodies. 2024; 13(1):11. https://doi.org/10.3390/antib13010011
Chicago/Turabian StyleAjay, Amrendra K., Martin Gasser, Li-Li Hsiao, Thomas Böldicke, and Ana Maria Waaga-Gasser. 2024. "TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth" Antibodies 13, no. 1: 11. https://doi.org/10.3390/antib13010011