Analysis of FA turnover by live-cell imaging

For FA turnover assay, coverslips with cells transfected by EGFP-target paxillin were then loaded onto the Dunn chamber and cells were observed every 30 s using 63 × objective by Leica DMI 6000B for a period of 1 h at 37 °C. The laser was supplied by EL6000 external light source. For EGFP images and FITC images, the 488-nm laser was used, and for TRITC images, the 561-nm laser was used.

FA turnover analysis was as described previously^{42, 56}. In brief, the background-subtracted fluorescent intensities of individual EGFP-paxillin inclusive adhesions in cells of varying differentiation states were measured over time using Image Pro-Plus software (Image Pro-Plus 6.0; Media Cybernetics, Silver Spring, MD). To measure the rate constant, the assembly (increasing fluorescence intensity) and disassembly (decreasing fluorescence intensity) period of FAs were plotted on separate semi-logarithmic graphs representing fluorescence intensity ratios in every minute. The apparent rate constant of assembly and disassembly were determined by the calculation of the slopes of linear regression trend lines fitted to the semi-logarithmic plots, which is detailed introduced in Fig. ^S5. For each rate, measurements were made on at least 10 individual adhesions in 5 separate cells of every differentiation state.