Fig. 9

Silencing of EFR3 gene expression affects EGF-induced Ca²⁺ influx. Fluo-4 Direct calcium assay kit was used according to the manufacturer’s protocol. First, cells were analyzed without EGF. Next, EGF was added to the same wells to the final concentration of 50 ng/ml. Fluorescence was acquired using a STELLARIS 8 system with a thermostated chamber at 37 °C and at excitation of 488 nm and emission within the range of 492–577 nm. Objective HC PL APO 86x/1.20 water numerical aperture 1.2 was used. Images were acquired every 1.3 s for 5 min. All image processing was performed using ImageJ Software (NIH). The fluorescence intensity of individual cells was obtained by defining a region of interest for each individual cell. The linear intensities were acquired from the “plot profile” over the entire 200 frames of the video. For further details see Methods and Additional file 2: Movie 1, Additional file 3: Movie 2