Applied precision

Live imaging was performed at room temperature following the method described by Fan et al. (2020 (link)) using the inverted wide‐field fluorescence system DeltaVision Core from Olympus AppliedPrecision (GE Healthcare Life Sciences) equipped with a 100×/1.4 UPlanSApo oil objective (Olympus) with an EM‐CCD camera. Immersion oil RI 1.514 (Zeiss) was used. Images and z‐sections for quantification of intracellular compartments and secretion were captured with 0.3 μm spacing using the SoftWoRx 5.5 software package (GE Healthcare Life Sciences) followed by deconvolution using the Resolve 3D‐constrained iterative deconvolution algorithm. Fixed tissues were imaged with an upright laser scanning confocal Zeiss LSM880. A Zeiss Plan Apochromat oil differential interference contrast objective 63×, NA 1.4 Plan APO oil DIC objective (Carl Zeiss) was used with RI 1.514 immersion oil (Zeiss). Z‐sections and images for quantification of intracellular compartments were captured with 0.5 μm spacing using the Zen Blue suite software according to previously published protocols (Corrigan et al., 2014 (link)). As previously reported (Prince et al., 2019 (link); Fan et al., 2020 (link)), fixation disrupts SC subcellular morphology, when compared to imaging of living ex vivo glands.

Expression plasmids containing full length and fragments of the β-catenin-GFP coding sequence under the control of the ADH1 promoter were transformed into the W303, Heh2-mCherry::NAT (BWCPL1314) strain. A yeast colony that incorporated the plasmid sequence was cultured and mounted onto a coverslip for live imaging on a DeltaVision wide-field microscope (GE Healthcare) with a CoolSnapHQ² CCD camera. Yeast fluorescent images were deconvolved using the iterative algorithm sofWoRx. 6.5.1 (Applied Precision, GE Healthcare). β-catenin-GFP transfected HeLa or HEK293T cells were mounted on Pro-Long Gold coated coverslip for imaging on a Zeiss Axio Observer microscope. All fluorescent images were analyzed with Fiji software. For quantification, the oval selection tool was used to draw a circle in both the nuclear and cytosolic regions on the same image plane to measure florescence intensity.

Cells were prepared according to the immunofluorescence protocol detailed above. Three-dimensional SIM imaging was performed on Deltavision OMX V4 microscope with an Olympus ×60 NA 1.42 objective and three PCO.edge sCMOS cameras and 488 nm, 568 nm and 647 nm laser lines. Cells were illuminated with a grid pattern and for each image plane, 15 raw images (5 phases and 3 orientations) were acquired. Super-resolution images were reconstructed from the raw image files and aligned using softWoRx software (Applied Precision, GE Healthcare). Images were processed in ImageJ/Fiji. 3D reconstructions were performed using Imaris.