Fig. 2 | Nature Communications

Fig. 2

From: A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis

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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