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Measured Effects of Wnt3a on Proliferation of HEK293T Cells Depend on the Applied Assay


Affiliations
1 University of Applied Sciences Kaiserslautern, Molecular Biology, Amerikastrasse 1, 66482 Zweibrucken, Germany
2 PHAST GmbH, Entenmuhlstrasse 48, 66424 Homburg, Germany
 

The Wnt signaling pathway has been associated with many essential cell processes. This study aims to examine the effects of Wnt signaling on proliferation of cultured HEK293T cells. Cells were incubated withWnt3a, and the activation of theWnt pathway was followed by analysis of the level of the β-catenin protein and of the expression levels of the target genes MYC and CCND1.The level of β-catenin protein increased up to fourfold.While the mRNA levels of c-Myc and cyclin D1 increased slightly, the protein levels increased up to a factor of 1.5. Remarkably, MTT and BrdU assays showed different results when measuring the proliferation rate ofWnt3a stimulated HEK293T cells. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the appliedWnt3a concentration. Oppositely, this correlation could not be shown in the MTT assays. The MTT results, which are based on the mitochondrial activity, were confirmed by analysis of the succinate dehydrogenase complex by immunofluorescence and by western blotting. Taken together, our study shows that Wnt3a activates proliferation of HEK293 cells.These effects can be detected by measuring DNA synthesis rather than by measuring changes of mitochondrial activity.
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  • Measured Effects of Wnt3a on Proliferation of HEK293T Cells Depend on the Applied Assay

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Authors

Patricia Reischmann
University of Applied Sciences Kaiserslautern, Molecular Biology, Amerikastrasse 1, 66482 Zweibrucken, Germany
Johanna Fiebeck
University of Applied Sciences Kaiserslautern, Molecular Biology, Amerikastrasse 1, 66482 Zweibrucken, Germany
Nadine von der Weiden
PHAST GmbH, Entenmuhlstrasse 48, 66424 Homburg, Germany
Oliver Muller
University of Applied Sciences Kaiserslautern, Molecular Biology, Amerikastrasse 1, 66482 Zweibrucken, Germany

Abstract


The Wnt signaling pathway has been associated with many essential cell processes. This study aims to examine the effects of Wnt signaling on proliferation of cultured HEK293T cells. Cells were incubated withWnt3a, and the activation of theWnt pathway was followed by analysis of the level of the β-catenin protein and of the expression levels of the target genes MYC and CCND1.The level of β-catenin protein increased up to fourfold.While the mRNA levels of c-Myc and cyclin D1 increased slightly, the protein levels increased up to a factor of 1.5. Remarkably, MTT and BrdU assays showed different results when measuring the proliferation rate ofWnt3a stimulated HEK293T cells. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the appliedWnt3a concentration. Oppositely, this correlation could not be shown in the MTT assays. The MTT results, which are based on the mitochondrial activity, were confirmed by analysis of the succinate dehydrogenase complex by immunofluorescence and by western blotting. Taken together, our study shows that Wnt3a activates proliferation of HEK293 cells.These effects can be detected by measuring DNA synthesis rather than by measuring changes of mitochondrial activity.