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To establish a simple and quantitative live cell fusion assay for placental syncytialization, we generated stable GFP and dsRed expressing fusogenic BeWo cell lines. Fluorescent Activated Cell Sorting was shown to provide a quantitative determination of forskolin (cAMP-mediated) fusion in a time and concentration dependent manner consistent with the increased secretion of beta human chorionic gonadotrophin (β-HCG) and appearance of multi-nucleated cells. Analyses of the fusion process demonstrated that in addition to increased cAMP levels, simultaneous reduction of intracellular calcium and inhibition of Type 1 phosphatidylinositol 3 kinase (PI3K)/Akt signaling also resulted in cell fusion. Although individual blockade of calcium channel function or PI3K/Akt signaling was without effect, the combination with forskolin resulted in a potentiation of cell fusion. These data demonstrate syncytialization is a complex process that depends upon the regulation of distinct signaling inputs that function in concert with each other.

Original publication

DOI

10.1371/journal.pone.0029353

Type

Journal article

Journal

PLoS One

Publication Date

2012

Volume

7

Keywords

Blotting, Western, Calcium, Calcium Channels, Cell Fusion, Cell Line, Tumor, Choriocarcinoma, Chorionic Gonadotropin, beta Subunit, Human, Class I Phosphatidylinositol 3-Kinases, Colforsin, Cyclic AMP, Female, Flow Cytometry, Humans, Lentivirus, Microscopy, Confocal, Placenta, Polymerase Chain Reaction, Pregnancy, Proto-Oncogene Proteins c-akt