Glass bottom imaging dishes

Antibodies (and their source) against the following targets were used: fibronectin (BD Biosciences, #610077); paxillin (Abcam, ab32084); phospho-myosin light chain 2 (pThr18/pSer19; Cell Signaling Technology, #3674); myosin light chain 2 (Cell Signaling Technology, #3672); vinculin (hVin-1; Millipore-Sigma V9131); phospho-FAK (pTyr397; Cell Signaling Technology, #3283); FAK (C-20; Santa Cruz Biotechnology; sc-558); YAP1 (D8H1X; Cell Signaling Technology, #14074). DAPI (4',6-diamidino-2-phenylindole dihydrochloride), Hoechst 33342, and phalloidins conjugated to Alexa Fluor 488, 594, or 647 were from ThermoFisher Scientific. Fasudil (also known as HA-1077) was from Cayman Chemical, PF-562271 was from ChemScene, and Blebbistatin (#203389) and latrunculin A (L5163) were from Millipore Sigma. Acrylamide and N,N-methylenebisAcrylamide were purchased from National Diagnostics. Human plasma-derived fibronectin (Corning #356008) was from Thermo Fisher. Glass-bottom imaging dishes were from Cellvis. Other chemicals and reagents, unless otherwise noted, were purchased from Millipore Sigma.

HEK293 cells stably expressing CGB-GFP and truncation mutants of CGB were plated on glass-bottom imaging dishes from Cellvis. Cells were induced from protein expression by the addition of doxycycline monohydrate (1 µg/ml; LKT Laboratories) for 10 h following which cells were fixed using 4% paraformaldehyde (Electron Microscopy Sciences) and co-stained with DAPI to label the nuclei. Cells were imaged on the Zeiss 880 confocal microscope. The bottom plane closest to the coverslip was imaged using a 63×/1.4 oil objective at room temperature. In some cells expressing GFP-tagged C-terminal portion of CGB, we observed signal from the nucleus and these cells were excluded from imaging.