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. 2017 Apr 19;7:908. doi: 10.1038/s41598-017-00955-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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(a) Schematic diagram of a multiplex assay system with two pcDNA3.1(+) vectors encoding molecular tension probe 2.4 (TP2.4) and ER LBD-RLuc8-SH2 (ERS), respectively. After expression, TP2.4 and ERS coexist in mammalian cells, which are ready to illuminate ligand activity. The working mechanisms of TP2.4 and ERS are briefly illustrated in the box-highlighted Switches 1 and 2. Upon stimulation by a ligand, TP2.4 or ERS exerts an intramolecular protein-to-protein interaction (PPI) (Switch 1). This PPI enhances the sandwiched luciferase’s activity. The enhanced activities are pinpoint visualized with specific coelenterazine (CTZ) analogues (Switch 2). (b) Specific optical intensities of the substrates in the multiplex assay system carrying two luciferase-based probes. Conventional assays are unable to discriminate the two optical signals. However, the present multiplex system pinpoint-illuminated the rapamycin or estrogen antagonist (OHT) activities without any signal contamination. Vehicles 1 and 2 are negative controls with 0.1% alcohol and 0.1% DMSO, respectively. Inset a briefly illustrates the experimental procedure with the specific substrates. The live cells were firstly stimulated with rapamycin and estrogen, lysed, and pinpoint-illuminated with a specific luciferin. Abbreviations: FRB, the rapamycin-binding domain of mTOR; FKBP, FK506-binding protein; ER LBD, the ligand binding domain of human estrogen receptor; SH2, the SH2 domain of v-Src; Rapa, rapamycin; DHT, 5α-dihydrotestosterone; OHT, 4-hydroxytamoxifen; E2, 17β-estradiol.