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CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

Tumor necrosis factor (TNF) is known to activate the epithelial Na(+) channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induc...

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Autores principales: Shabbir, Waheed, Topcagic, Nermina, Aufy, Mohammed, Oz, Murat
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917654/
https://www.ncbi.nlm.nih.gov/pubmed/33673381
http://dx.doi.org/10.3390/ijms22041858
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author Shabbir, Waheed
Topcagic, Nermina
Aufy, Mohammed
Oz, Murat
author_facet Shabbir, Waheed
Topcagic, Nermina
Aufy, Mohammed
Oz, Murat
author_sort Shabbir, Waheed
collection PubMed
description Tumor necrosis factor (TNF) is known to activate the epithelial Na(+) channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na(+) current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na(+) current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.
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spelling pubmed-79176542021-03-02 CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells Shabbir, Waheed Topcagic, Nermina Aufy, Mohammed Oz, Murat Int J Mol Sci Article Tumor necrosis factor (TNF) is known to activate the epithelial Na(+) channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na(+) current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na(+) current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC. MDPI 2021-02-12 /pmc/articles/PMC7917654/ /pubmed/33673381 http://dx.doi.org/10.3390/ijms22041858 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Shabbir, Waheed
Topcagic, Nermina
Aufy, Mohammed
Oz, Murat
CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells
title CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells
title_full CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells
title_fullStr CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells
title_full_unstemmed CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells
title_short CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells
title_sort crispr/cas9 mediated knock down of δ-enac blunted the tnf-induced activation of enac in a549 cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917654/
https://www.ncbi.nlm.nih.gov/pubmed/33673381
http://dx.doi.org/10.3390/ijms22041858
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AT topcagicnermina crisprcas9mediatedknockdownofdenacbluntedthetnfinducedactivationofenacina549cells
AT aufymohammed crisprcas9mediatedknockdownofdenacbluntedthetnfinducedactivationofenacina549cells
AT ozmurat crisprcas9mediatedknockdownofdenacbluntedthetnfinducedactivationofenacina549cells