The TIRF experiments were done on a Nikon Ti motorized inverted microscope with Perfect Focus System, 491-nm laser (50 mW max power) with 50-ms exposure time. Images or movies were acquired with MetaMorph software. The microscope has temperature and CO2 level control to minimize the environment perturbation on cells.
The cells were either grown on γ-irradiated 35-mm glass bottom dishes (MatTek P35G-0.170-14-C) or on high-precision microscope cover glasses (Marienfeld 0117650 lot. 33825 819). The glass was coated with 10 μg/mL fibronectin (Sigma-Aldrich F0895) in PBS for 10 min before cells were plated. A total of 100 nM LGK974 (Caymanchem 14072) was added to the cell culture medium when the cells were plated to decrease the autocrine Wnt signaling. The cells were cultured in normal DMEM and switched into FluoroBrite (Thermo Fisher A1896701) with 10% FBS and 100 nM LGK974 before imaging. The imaging experiments were normally done the next day after the cells were plated.
The cells were either grown on γ-irradiated 35-mm glass bottom dishes (MatTek P35G-0.170-14-C) or on high-precision microscope cover glasses (Marienfeld 0117650 lot. 33825 819). The glass was coated with 10 μg/mL fibronectin (Sigma-Aldrich F0895) in PBS for 10 min before cells were plated. A total of 100 nM LGK974 (Caymanchem 14072) was added to the cell culture medium when the cells were plated to decrease the autocrine Wnt signaling. The cells were cultured in normal DMEM and switched into FluoroBrite (Thermo Fisher A1896701) with 10% FBS and 100 nM LGK974 before imaging. The imaging experiments were normally done the next day after the cells were plated.