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PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression
The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from PKN2(flox/flox) mouse embryos. PKN2(flox/flox) cells formed cell aggregates in flat bottom low attachment well pla...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787963/ https://www.ncbi.nlm.nih.gov/pubmed/33437883 http://dx.doi.org/10.1016/j.bbrep.2020.100895 |
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author | Kubouchi, Koji Mukai, Hideyuki |
author_facet | Kubouchi, Koji Mukai, Hideyuki |
author_sort | Kubouchi, Koji |
collection | PubMed |
description | The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from PKN2(flox/flox) mouse embryos. PKN2(flox/flox) cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre;PKN2(flox/flox) cells in which PKN2 was depleted by the introduction of Cre-recombinase rarely formed aggregates. Time-lapse analysis revealed that the velocity of Cre;PKN2(flox/flox) cell motility was significantly lower than that of PKN2(flox/flox) in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre;PKN2(flox/flox) cells. Conversely, Cre;PKN2(flox/flox) cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre;PKN2(flox/flox) cells with decreased roundness, although PKN2(flox/flox) cells underwent compaction in a round shape spheroid within 24 h. Immunoblot analysis revealed that the preparation of the cell suspension from adherent conditions using trypsin/EDTA treatment significantly decreased the expression of N-cadherin in both PKN2(flox/flox) and Cre;PKN2(flox/flox) cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre;PKN2(flox/flox) cells compared to PKN2(flox/flox) cells. Reverse transcription quantitative PCR revealed that N-cadherin mRNA in Cre;PKN2(flox/flox) cells was significantly lower than that of PKN2(flox/flox) cells. These results suggest that PKN2 controls the velocity of cell motility and the transcription of N-cadherin in fibroblasts, leading to cell aggregation and compaction for spheroid formation in suspension culture. |
format | Online Article Text |
id | pubmed-7787963 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-77879632021-01-11 PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression Kubouchi, Koji Mukai, Hideyuki Biochem Biophys Rep Short Communication The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from PKN2(flox/flox) mouse embryos. PKN2(flox/flox) cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre;PKN2(flox/flox) cells in which PKN2 was depleted by the introduction of Cre-recombinase rarely formed aggregates. Time-lapse analysis revealed that the velocity of Cre;PKN2(flox/flox) cell motility was significantly lower than that of PKN2(flox/flox) in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre;PKN2(flox/flox) cells. Conversely, Cre;PKN2(flox/flox) cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre;PKN2(flox/flox) cells with decreased roundness, although PKN2(flox/flox) cells underwent compaction in a round shape spheroid within 24 h. Immunoblot analysis revealed that the preparation of the cell suspension from adherent conditions using trypsin/EDTA treatment significantly decreased the expression of N-cadherin in both PKN2(flox/flox) and Cre;PKN2(flox/flox) cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre;PKN2(flox/flox) cells compared to PKN2(flox/flox) cells. Reverse transcription quantitative PCR revealed that N-cadherin mRNA in Cre;PKN2(flox/flox) cells was significantly lower than that of PKN2(flox/flox) cells. These results suggest that PKN2 controls the velocity of cell motility and the transcription of N-cadherin in fibroblasts, leading to cell aggregation and compaction for spheroid formation in suspension culture. Elsevier 2021-01-02 /pmc/articles/PMC7787963/ /pubmed/33437883 http://dx.doi.org/10.1016/j.bbrep.2020.100895 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Short Communication Kubouchi, Koji Mukai, Hideyuki PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression |
title | PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression |
title_full | PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression |
title_fullStr | PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression |
title_full_unstemmed | PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression |
title_short | PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression |
title_sort | pkn2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and n-cadherin expression |
topic | Short Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787963/ https://www.ncbi.nlm.nih.gov/pubmed/33437883 http://dx.doi.org/10.1016/j.bbrep.2020.100895 |
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