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. 2018 Mar 13;9:1061. doi: 10.1038/s41467-018-03278-w

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© The Author(s) 2018

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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Fig. 2 — LATS-BS can be used to measure LATS activity in vitro and in vivo. a LATS-BS can be used to assess LATS activity in different cell lines. LATS-BS was transfected alone or together with LATS2-FLAG into HEK293, MDA-MB231, or A549 cells. Biosensor activity was measured by luciferase assay or BLI of live cells (n = 3). For BLI, data were heat-mapped (red signal denotes an increase in LATS-BS activity, whereas blue indicates a decrease). b The LATS-BS responds to cell confluency. LATS-BS or pGL3-control were transfected into HEK293 at different confluencies. After 48 h, biosensor activity was determined by luciferase assay using cell lysate (top) or bioluminescent imaging (BLI) of live cells (bottom) (n = 3). c, d LATS-BS is inhibited by LPA (c) and activated by forskolin (d). LATS-BS or pGL3-control were transfected into HEK293. Cells were treated with increasing concentrations of LPA for 1 h or with forskolin for 30 min before luciferase assay and BLI (n = 3). e LATS-BS responds to drug treatments regulating Hippo signaling. HEK293 were transfected with LATS-BS and treated with the following: PI3K inhibitor 1 (GDC0941), 10 μM for 4 h; PDK inhibitor (GSK2334470), 10 μM for 4 h; EGF, 100 ng ml⁻¹ for 1 h; Insulin, 10 μg ml⁻¹ for 1 h; F/IBMX (Forskolin/IBMX), 10 μM Forskolin/100 μM IBMX for 1 h; PI3K inhibitor 2 (LY294002), 10 μM for 4 h; LPA, 10 μM for 1 h; Sphin gosine1-phosphophate (S1P), 1 μM for 1 h; 12-O-tetradecanoylphorbol-13-acetate (TPA), 5 nM for 1 h; 2-deoxy glucose, 25 mM for 1 h. Biosensor activity was determined by luciferase assay or BLI of live cells (n = 3). f, g LATS-BS can be used to observe LATS activity under the microscope. LATS-BS was stably overexpressed in A549 and MDA-MB231. Cells were imaged using LV200 BLI. Images in g are higher magnification of images in f. Scale bar represents 100 μm (f) or 20 μm (g). h LATS-BS can be used to determine LATS activity in vivo. HEK293 were transfected with LATS-BS. Cells were injected into the mammary fat pad of immunocompromised mice. After 48 h, BLI was performed. Data are represented as mean ± SD

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