HEK293, HPAF-II, and PANC-1 cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% l-glutamine (all from Invitrogen Technologies) in a humidified CO2 incubator at 37°C. Gαq/11 KO HEK293 cells were a gift from A. Inoue (Tohoku University, Japan) (45). All cell culture plastics were from Sarstedt, unless otherwise specified. For dcFRAP experiments, cells were seeded on 35-mm ECM gel–coated (1:300; Sigma-Aldrich) glass-bottom dishes (Greiner Bio-One four-compartment 35-mm glass-bottom dishes). Cells were transfected with Lipofectamine 2000 24 hours before analysis. For FRET efficiency measurements and FZD-mediated DVL recruitment, cells were seeded onto round 24-mm coverslips precoated with poly-d-lysine in six-well plates. Cells were transfected 4 to 5 hours later using the Effectene transfection reagent (Qiagen). Cell culture medium was replaced 16 to 18 hours later, and the analysis was done 48 hours after transfection. To investigate the cellular expression of the receptor sensors or their FRET efficiency, 500 ng of DNA per well of each sensor was used for transfection. HEK293 cells stably expressing the sensor V5-FZD5-FlAsH436-CFP were also used for the experiments following the same procedure. For FRET efficiency control experiments, cells were cotransfected with 300 ng of V5-FZD5-CFP and 300 ng of V5‑FZD5-FlAsH. Pharmacological inhibition of Gαq signaling was accomplished with structurally related compounds 1 μM FR900359 (64) or 100 nM YM-254890 (Wako Laboratory Chemicals). PKC inhibition was achieved with 100 nM Gö 6983 (Sigma-Aldrich). C59 was used to inhibit PORCN to reduce endogenous secretion of WNTs (65). For stimulation, recombinant WNT-5A (645-WN; R&D Systems/Bio-Techne) was used.

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