Fluorescence imaging of FZD5 FRET sensors was performed as previously described (25). Briefly, 5 mM of BAL was used to determine the FRET efficiency of the constructs. This compound removes FlAsH from its binding motif in the receptor, resulting in a dequenching of CFP fluorescence. Coverslips with the cells were mounted using an Attofluor holder (Molecular Probes), and the cells were maintained in imaging buffer. BAL was added to the cells after 20 to 30 s. Recovery of CFP fluorescence was monitored over time, and FRET efficiency was calculated by inputting the minimum and maximum values of CFP into the following equation: ΔE/Emax × 100. Fluorescence intensities data were acquired using Clampex software (Axon Instruments). The FRET ratio is calculated as the fluorescence intensity of FlAsH over that of CFP and subsequently corrected for the signal obtained in vehicle-treated cells.

To investigate the activation of the receptor in single cells, HEK293 cells stably expressing the receptor sensor V5‑FZD5-FlAsH436-CFP were seeded onto WillCo-dish 40-mm glass-bottom dishes precoated with poly-d-lysine. For G protein activation studies, HEK293 cells were seeded onto these plates and transfected 3 to 4 hours later using the Effectene reagent (Qiagen). For transfection, 600-ng DNA of FZD5 and 200-ng DNA of the G protein FRET sensor were used. Culture medium was replaced 16 to 18 hours later, and cells were incubated overnight with the PORCN inhibitor LGK-974 (Cayman Chemical) at a final concentration of 0.1 μM. Analysis was carried out 48 hours after seeding the cells. To monitor receptor activation, FlAsH labeling of the receptor sensors was performed. Cells were maintained in imaging buffer. The BioPen microfluidic system (Fluicell) was used to deliver the ligands. Recombinant WNT-5A (R&D Systems) was dissolved in imaging buffer containing 0.1% bovine serum albumin (BSA). Saturating concentrations of WNT-5A were used for single-cell experiments: 2000 ng/ml for receptor activation and 1000 ng/ml for G protein activation. Fluorescence intensities data were acquired using Clampex software (Axon Instruments).

To investigate the activation of G proteins in populations of cells, HEK293 cells were seeded in 100-mm plates, and 48 hours later, with a 60 to 70% confluence, cell culture medium was exchanged, and cells were transfected using the Effectene reagent (Qiagen) according to the manufacturer’s instructions. For transfection, the following DNA was used per plate: 1.8 μg of V5-FZD5 or pcDNA3 and 600 ng of the G protein FRET sensor. Twenty-four hours after transfection, black 96-well BRANDplates, flat bottom, were coated for 30 min using poly-d-lysine. After washing with sterile PBS, 30,000 cells were seeded per well. To investigate the activation of the receptor, stable cells expressing the receptor sensor V5-FZD5-FlAsH436-CFP were seeded in 100-mm plates, and 30,000 cells per well were placed in 96-well plates 72 hours later. Analysis of the cells was done 24 hours after seeding the cells in the 96-well plates using Synergy Neo2 Multi-Mode Microplate Reader (BioTek), with Gen5 Data Analysis Software. Cells were excited at 420 nm, and emission was detected at 485/540 nm. During measurements, cells were maintained at 37°C in imaging buffer containing 0.1% BSA. Recombinant WNT-5A was added to the cells 5 min after the reading started. Fluorescence changes were recorded for an additional 20 min. Data were analyzed using the software GraphPad Prism 6.

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