Csu w1

siControl and siMASTL MDA-MB-231 cells silenced for 48 h expressing mEmerald-paxillin were plated on ibidi plate (80826) coated with collagen (5 µg/ml) for 3 h before imaging. Fluorescent cells were then imaged live using a Marianas spinning disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 (Intelligent Imaging Innovations). Images were acquired every minute using a Photometrics Evolve, 10-MHz back-illuminated electron multiplying charge-coupled device (512 × 512 pixels) camera and 40× Zeiss long distance C-Apochromat water objective (NA 1.1).
To quantify focal adhesion dynamics, videos were preprocessed for brightness and background with ImageJ and further processed using the Focal Adhesion Analysis Server (http://faas.bme.unc.edu/; Berginski and Gomez, 2013 (link)) with the following analysis settings: minimum (min) adhesion size: 1; focal adhesion (FA) phase length: 5; and focal adhesion alignment index (FAAI): 3.

Fixed and stained Caco-2 cells were captured on a Hamamatsu sCMOS Orca Flash4.0 V2 using a 3i Marianas CSU-W1 spinning-disk confocal microscope using a 100× Zeiss Plan-Apochromat NA 1.4 and Z step size 270 nm. DNA-DAPI was imaged with a 405 nm laser and 445/45 nm filter, lamin A–Alexa 488 imaged with a 488 nm laser and 525/30 nm filter, and Keratin 8–Alexa 568 imaged with a 561 nm laser and 617/73 nm filter. The image was deconvolved with Scientific Volume Imaging Huygens Essential version 21.04 using a theoretical PSF and the CMLE algorithm, with SNR:12 and max 40 iterations. The video was created using Arivis Vision4D version 3.5.