Live cell imaging

Cells were transfected, incubated for 24 hr, and re-plated prior to imaging on 10 μg/ml fibronectin–coated glass-bottomed dishes (MatTek Corporation). The time-lapse images were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), with an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner, or with an inverted microscope (IX-71; Olympus) equipped with a Polychrome IV monochromator (TILL Photonics). Both systems have appropriate filters, heated sample chamber (+37°C), and controlled CO2. With 3I Marianas, a 63x/1.2 W C-Apochromat Corr WD = 0.28 M27 objective was used. SlideBook 5.0 software (3I intelligent Imaging Innovations) and sCMOS (Andor) Neo camera were used for the image acquirement and recording. With Olympus, a 60x water objective with 1.6× magnification was used. TILL Vision 4 software (TILL Photonics) and Imago QE (TILL Photonics) and Andor iXon (Andor) cameras were used for the image acquirement and recording. Deconvolution of the time-lapse videos was performed with AutoQuant AutoDeblur 2D non-blind Deconvolution (AutoQuant Imaging, Inc.). Further analyses of the video frames were performed with Image Pro Plus 6.0.