Fibronectin coated

About 5.3 × 10⁵ cells/ml were seeded in each side of a silicon insert, previously adhered to a well of a μ-Slide 8 Well Collagen IV or Fibronectin coated (IBIDI). Cells were kept in culture for 24 h at 37 °C in 5% CO₂ atmosphere conditions. The inserts were then removed, cells were washed with RPMI1640 culture medium supplemented with 10% FBS, and fresh supplemented medium was added to each well. Time-lapse microscopy was performed using Leica DMI6000 (Wetzlar) and three bright field images per well/condition were acquired for 24 h with intervals of 10 min. The wound healing rate was evaluated by measuring the total wound area at each time point using the ImageJ software (https://imagej.nih.gov/ij/). Migration assay results were depicted as the average values of the percentage of closing wound + SD. The percentage of closing wound was calculated by subtracting the area of the open wound at the first time point (t = 0 h) to the area determined for each time point, followed by normalization of the resulting values to the wound area determined for the first time point (t = 0). Two independent biological experiments with at least technical triplicates were analyzed.

For TIRFM, AECs were grown on fibronectin-coated μ-slide 8 well glass bottom slides (Ibidi) for 4 days and stained with 5 μl/ml Vybrant DiO cell labeling solution (Thermofisher) for 10 min at 37 °C. Life imaging was performed in the imaging facility of the Molecular Biophysics group, HU Berlin on a Confocal Laser Scanning Microscope (Olympus FV-1000MPE) equipped with a TIRFM upgrade at 37 °C and 5% CO₂ using a 63x oil objective and a cooled CCD camera. Images were taken at indicated time points and analysed using FijI. Particle analysis was performed from binary images after thresholding (method Isodata). Total intensity of attachment from all detected particles was measured.