CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins
Abstract
:1. Introduction
2. Results
2.1. The CAIX Protein Distributes to Proteolytically Active Invadopodia
2.2. Bicarbonate Transporter NBCe1 Colocalizes with Cortactin and CAIX in Invadopodia
2.3. Suppression of CAIX Reduces Invadopodia Formation
2.4. Loss of CAIX Decreases Levels of Invadopodia Components and Signaling
2.5. Treatment of Tumor Cells with CAIX Targeting Antibodies Suppresses Their Metastatic Properties
2.6. Effect of anti-CAIX Antibody Treatment on Experimental Lung Metastasis of HT1080 Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Transient Silencing
4.3. Immunoblotting
4.4. Stable Transfection
4.5. Immunofluorescence
4.6. Cell Fractioning
4.7. Quantification of Invadopodia Formation via Cortactin Staining
4.8. Cell Invasion Assay
4.9. Matrigel Degradation Assay
4.10. Quail Chorioallantoic Membrane (CAM) Model
4.11. Tumor Cells Implantation
4.12. Immunohistochemistry
4.13. In Vivo Model of Experimental Metastasis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Antigen | Host | Methodology–Dilution | Company |
---|---|---|---|
actin | g | WB - 1:1000 | Santa Cruz |
anion exchanger 2 (AE2) | r | IF - 1:500 | GeneScript–on request [14] |
Arp2 | r | WB - 1:1000 | Santa Cruz (sc-15389) |
carbonic anhydrase IX (CAIX) | m | WB - 1:3 (hybridoma medium) IF - 1:250 (M75-AlexaFluor 488 conjugate) IHC - 1:100 | in house, M75 antibody [9] |
cortactin | m | WB - 1:1000 IF - 1:100 | Millipore (05-180) |
F-actin (phalloidin AF-555) | - | IF - 1:40 | Invitrogen (A34055) |
integrin beta 3 | r | WB - 1:1000 | Abcam (ab75872) |
Ki-67 | m | IHC - 1:100 | DAKO (M7240) |
matrix metalloproteinase 14 (MMP14) | r | WB - 1:1000 | Millipore (AB6004) |
electrogenic sodium bicarbonate cotransporter 1 (NBCe1) | r | IF - 1:100 | Millipore (AB3212) |
paxillin | r | IF - 1:250 | Santa Cruz (sc-5574) |
phospho-cortactin | r | WB - 1:1000 | Cell Signaling (4569) |
phospho- protein-kinase A (p-PKA) | r | WB - 1:1000 | Cell Signaling (5661S) |
protein-kinase A (PKA) | r | WB - 1:1000 | Cell Signaling (4782S) |
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Debreova, M.; Csaderova, L.; Burikova, M.; Lukacikova, L.; Kajanova, I.; Sedlakova, O.; Kery, M.; Kopacek, J.; Zatovicova, M.; Bizik, J.; et al. CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins. Int. J. Mol. Sci. 2019, 20, 2745. https://doi.org/10.3390/ijms20112745
Debreova M, Csaderova L, Burikova M, Lukacikova L, Kajanova I, Sedlakova O, Kery M, Kopacek J, Zatovicova M, Bizik J, et al. CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins. International Journal of Molecular Sciences. 2019; 20(11):2745. https://doi.org/10.3390/ijms20112745
Chicago/Turabian StyleDebreova, Michaela, Lucia Csaderova, Monika Burikova, Lubomira Lukacikova, Ivana Kajanova, Olga Sedlakova, Martin Kery, Juraj Kopacek, Miriam Zatovicova, Jozef Bizik, and et al. 2019. "CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins" International Journal of Molecular Sciences 20, no. 11: 2745. https://doi.org/10.3390/ijms20112745