For live-cell imaging experiments, A549 were plated onto six-well plastic plates (cell division assay) or glass-bottomed dishes (CAR junctional analysis and microtubule tracking) (Ibidi). For cell division assays, Hepes (25 mM) was added to the cells that were then imaged every 5 min for 12 hours using phase-contrast imaging and 488- and 568-nm laser excitation using a 20× objective on an Olympus IX71 inverted fluorescence microscope equipped with a humidified environmental chamber heated to 37°C. All images were saved as avi files. For CAR junctional analysis, cells were imaged every minute using 488-nm laser excitation using a 60× oil objective on a Nikon A1R inverted confocal microscope (Nikon) equipped with a humidified environmental chamber heated to 37°C, with perfect focus system activated. After 5 min, EGF was added to the imaging media at a final concentration of 10 ng/ml, and the imaging was immediately resumed for a further 60 min. All images were saved as nd2 files and analyzed in ImageJ or exported as tif files for presentation. For microtubule tracking assays, time-lapse movies of A549 cells expressing tubulin-GFP were acquired on an inverted Nikon A1R confocal laser scanning microscope using a 100× oil objective (numerical aperture, 1.45) at a rate of one frame per 2 s with or without EGF addition (10 ng/ml). Images were saved as nd2 files and analyzed in ImageJ or exported as tif files for presentation.

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