Alexa fluor 546 488

Similar number of cells were seeded on 10 μg/mL fibronectin-coated (Roche, USA) or 10 μg/mL laminin-coated (Sigma-Aldrich, USA) coverslips for 6 h to allow for single cell imaging. Samples were fixed with 4% formaldehyde (Sigma-Aldrich, USA) for 20 min at room temperature, permeabilized in 0.2% Triton-X-100 (Sigma-Aldrich, USA) for 10 min at room temperature, and blocked with 4% bovine serum albumin (Sigma-Aldrich, USA) for minimum 30 min. Cells were then incubated with primary antibodies in blocking buffer at 4 °C overnight, followed by secondary antibody incubation for 1 h at room temperature. Stress fibers were stained with phalloidin conjugated with Alexa Fluor 546/488 (Invitrogen, USA). Coverslips were mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, USA). Images were acquired using Zeiss LSM 710/980 confocal microscopes (Carl Zeiss, Germany) and analyzed with FIJI software (NIH, version 1.53)^{69 (link)}. Co-localization analysis was carried out with Coloc2 plugin in FIJI software (NIH, version 1.53c)^{69 (link)} with the segmented FA as the mask^{34 (link)}.

For immunofluorescence microscopy, the automated microscope Pathway 855 (Becton Dickinson, Franklin Lakes, NJ, United States) was used to read fluorescence intensity in 96-well plates. For confocal microscopy, LSM 510 laser scanning microscope (Carl Zeiss, Germany) was used.
The cells were fixed in 3.7% paraformaldehyde for 20 min, followed by permeabilization with 0.5% triton-X in PBS for 15 min and blocking for 15 min using blocking solution (3% BSA in PBS). The primary antibody to phospho-H2AX (05-636, Millipore)/phospho-Rad17 (6981, Cell Signaling Technology)/CtIP (61142, Active Motif), diluted in blocking solution, was added for 1 h, followed by incubation with a secondary antibody (Alexa-Fluor 546/488) and Hoechst 33342 (Invitrogen) diluted in blocking solution for 45 min.
Images were captured and analyzed using the BD Pathway software, wherein the region of interest (ROI), in this case the cell nuclei, were defined by Hoechst stain, and the average intensity of the antibody-coupled fluorescence within each ROI was determined.