Adherens Junction Dynamics Regulation

Cells were seeded on coverslips and grown to confluence, then cells were either treated with standard medium, starved for 8 h, or treated with 5 μM Rapamacyn for 24 h or 10 mM 4-dithiothreitol (DTT; Sigma-Aldrich) for 3 h. Immunofluorescence staining was then carried out as above described. Alexa Fluor 488 or 594 Phalloidin (Thermo Fisher Scientific) was used to probe F-actin. Images were captured with TCS-SP8 Confocal System (Leica Microsystems) and analyzed with Fiji/ImageJ software (Schneider et al., 2012 (link)), similarly, to what previously described (Di Russo et al., 2017 (link)). Briefly, adherens junctions, considered as the E-cadherin/beta-catenin positive cell-cell border fragment, were measured on the focal plane corresponding to the maximum intensity of the E-cadherin signal. Adherens junctions ratio was calculated as the length of adherens-junctions over the length of the perimeter of the cell that was manually measured by using the border of phalloidin maximum intensity projections. Experiments were performed in triplicate and, overall, at least 100 cells per sample were measured.
Time-lapse images of cells were captured every 20 min for 20 h by AF6000 microscope system and LASX software (Leica Microsystems).