Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying

Authors

Alejandro Sarrion-Perdigones
Lyra Chang
Yezabel Gonzalez
Tatiana Gallego-Flores
Damian W Young
Koen J T Venken

Publication Date

6-1-2020

Journal

Current Protocols in Molecular Biology

DOI

10.1002/cpmb.122

PMID

32539239

PMCID

PMC7806208

Published Open-Access

no

Keywords

A549 Cells, Escherichia coli, Genes, Reporter, Genetic Vectors, Humans, Luciferases, Plasmids, Promoter Regions, Genetic, Recombinant Proteins, Signal Transduction, Transfection, Luciferase, assay, multiplex, hextuple, orthogonal, high-throughput, cellular signaling pathway, cell culture, transfection, pathway perturbation, plate reader

Abstract

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway, and a second that is coupled to a control pathway for normalization purposes. We expanded this approach to multiplex hextuple luciferase assaying that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters. Here, we present detaileded protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κβ, TGF-β, p53, and MAPK/JNK), using the constitutive CMV promoter as normalization control. Protocols are provided for preparing reporter vector plasmids for multiplex reporter assaying, performing cell culture and multiplex luciferase reporter vector plasmid transfection, executing multiplex luciferase assays, and analyzing and interpreting data obtained by a plate reader apppropriately equipped to detect the different luminescences. Protocols on how to tailor multiplex hextuple luciferase assaying to different cellular signaling pathways are explained in accompanying Current Protocols in Molecular Biology article (Sarrion-Perdigones et al., in press).

Basic Protocol 1:

Preparation of vectors for multiplex hextuple luciferase assaying

Basic Protocol 2:

Cell culture work for multiplex hextuple luciferase assays

Basic Protocol 3:

Transfection of luciferase reporter plasmids followed by drug and recombinant protein treatments

Basic Protocol 4:

Performing the multiplex hextuple luciferase assay

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