Journal: Nature Communications
Article Title: A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis
Figure Lengend Snippet: 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
Article Snippet: The following compound treatments were used for in vitro luciferase assay and live-cell imaging: RAF inhibitor (GW5054, Cayman Chemical), ATR inhibitor (CGK733, Cayman Chemical), PI3K inhibitor 1 (GDC0941, Cayman Chemical), PI3K inhibitor 2 (LY294002, Cayman Chemical), PDK inhibitor (GSK2334470, Cayman Chemical)—10 μM for 4 h, EGF- 100 ng ml−1 for 1 h; insulin (Sigma 91077 C)—10 μg ml−1 for 1 h; F/IBMX (Forskolin, Cayman Chemical /IBMX, Cayman Chemical)—0.1–10 μM for Forskolin and 100 μM for IBMX for 1 h; L-α–LPA (Sigma L7260)—0.1–10 μM for 1 h, S1P-1 μM for 1 h; TPA (Cell signaling #41745)—5 nM for 1 h; and 2-deoxy glucose (Sigma #D8375)—25 mM for 1 h. For the LV200 imaging, 3.5 mM d -luciferin was added to the media culturing HEK293, A549, or MDA-MB231 cells stably expressing LATS-BS at 5–10 min before imaging.
Techniques: Activity Assay, In Vitro, In Vivo, Transfection, Multiple Displacement Amplification, Luciferase, Imaging, Microscopy, Stable Transfection, Injection, Mouse Assay