Wnt Signaling Pathway Activation Assay

Tfr1 cells, the R1 subclone carrying the TOPflash luciferase reporter plasmid, were cultured and differentiated in the same manner as the maternal R1 ES cell line. For cell stimulation, recombinant WNT3A (R&D Systems), WNT3A conditioned media or control conditioned media^{20 (link)}, TPA, and CHIR99021 (Sigma-Aldrich) were added 12 h before harvesting. Luciferase assay kit (Promega, USA) was used according to the manufacturer's instructions for the evaluation of luciferase activity. Relative luciferase units were measured on a Chameleon V luminometer (Hidex, Finland) and normalized to the cell mass.

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Radaszkiewicz K.A., Beckerová D., Woloszczuková L., Radaszkiewicz T.W., Lesáková P., Blanářová O.V., Kubala L., Humpolíček P, & Pachernik J. (2020). 12-O-Tetradecanoylphorbol-13-acetate increases cardiomyogenesis through PKC/ERK signaling. Scientific Reports, 10, 15922.

Publication 2020

WNT3A CHIR99021 Luciferase assay kit Chameleon V luminometer

Assay Cell Chameleon Chir99021 Conditioned media Luciferase Maternal cell Plasmid Promega Tfr1

Corresponding Organization :

Other organizations : Masaryk University, Czech Academy of Sciences, Institute of Biophysics, Tomas Bata University in Zlín

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Variable analysis

independent variables

Recombinant WNT3A (R&D Systems)
WNT3A conditioned media
Control conditioned media
CHIR99021 (Sigma-Aldrich)

dependent variables

Luciferase activity

control variables

Cell culture and differentiation conditions were the same as the maternal R1 ES cell line

controls

Negative control: Control conditioned media

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