phases. The assembly and disassembly rates were varied by shortening or lengthening the amount of time each adhesion spent reaching its maximum intensity. The stability period in each of these adhesions was set to five frames. Assembly and disassembly lengths between 10 and 20 were all tested. In order to avoid biasing the automated assembly and disassembly phase fitting software to higher phase lengths, the minimum phase length was set to five time points during image analysis. Overall, the software was able to reliably extract both the expected assembly and disassembly rates and length of time spent in each phase (Figure S13). There were several samples in the longer phase lengths that were predicted to have substantially shorter assembly and disassembly phase lengths than that specified by the software, but these simulated adhesions were in the minority and did not significantly affect the confidence intervals around the mean assembly and disassembly lengths. These simulations further support the accuracy of results derived from applying the same sets of algorithms to the analysis of adhesions in living cells. Statistical Tests Two different types of tests were used to determine the statistical significance of the differences between the adhesions in the wild- type, S178A and labeled-FAK adhesions. To compare datasets with ,2000 points, bootstrap resampling was used to determine either the mean or median distribution. From these distributions the p-value was determined using the percentile method. The bootstrap method was too computationally intense to compare datasets, such as the area and axial ratio of the adhesions, with significantly more points than 2000 data points. Instead, the Wilcox Rank Sum test was used to find the p-value in these cases. Software Availability The most recent version of the software system is available from the Gomez lab website (http://gomezlab.bme.unc.edu/tools). In addition to the source code, released under the BSD license, there is a sample movie that can be used to test the success of installing the analysis system. The software has been tested on Mac OS610.5 and Ubuntu Linux 10.04. Supporting Information Figure S1 The assembly and disassembly log-linear mod- els fit the Paxillin intensity time courses with high R2 values. The red lines indicate the median length-adjusted R2 values. (PNG) Figure S2 Adhesions labeled with EGFP-FAK are larger in mean area and have a larger axial ratio than those labeled EGFP-Paxillin. There are 51836 adhesions in the FAK data set and 44685 adhesions in the Paxillin data set. The p-values were calculated using the same methods as Figure 6. (PNG) Figure S3 There are no significant differences between the expression levels in the EGFP-Paxillin and EGFP- PaxillinS178A cell lines. The average intensity of fluorescence inside the cell is shown in three different ways: the overall cell intensity (A), inside the cell not including the adhesions (B) and only the adhesions (C). The error bars are 95% confidence intervals determined using 50,000 bootstrap samples on the mean value. (PNG) Figure S4 Flow chart for the tracking software adhesion following algorithm. (PNG) Quantifying Focal Adhesion Dynamics Figure S5 Changing the adhesion detection threshold does not affect the differences in the assembly rates between S178A mutant and wild-type cells. Each boxplot contains all the adhesions with significant linear fits (linear model p-value below 0.05). The p-values in each boxplot are for the difference in medians between the wild-type and S178A data sets in each boxplot. (PNG) Figure S6 Changing the adhesion detection threshold does not affect the differences in the disassembly rates between S178A mutant and wild-type cells. Each boxplot contains all the adhesions with significant linear fits (linear model p-value below 0.05). The p-values in each boxplot are for the difference in medians between the wild-type and S178A data sets in each boxplot. (PNG) Figure S7 Changing the minimum length of the assem- bly phase does not significantly affect the differences in the assembly rate between the wild-type and S178A mutant cells. Each boxplot contains all the adhesions with significant linear fits (linear model p-value below 0.05). The p- values in each boxplot are for the difference in medians between the wild-type and S178A data sets in each boxplot. (TIFF) Figure S8 Changing the minimum length of the disas- sembly phase does not significantly affect the differenc- es in the assembly rate between the wild-type and S178A mutant cells. Each boxplot contains all the adhesions with significant linear fits (linear model p-value below 0.05). The 95% confidence intervals on the percent change in the median assembly rate between the wild-type and S178A adhesions overlap in all minimum length settings. The p-values in each boxplot are for the difference in medians between the wild-type and S178A data sets in each boxplot. (PNG) Figure S9 Reducing the time between each frame only has mild effects on the assembly and disassembly rates in the wild-type cells. The label ‘All’ indicates that none of the images were excluded to estimate the rates, while ‘Sampled’ indicates that every other image from each experiment was discarded. To compensate for the shortened experimental time, the minimum number of points needed to determine an assembly or disassembly rate was reduced to 5 for the sampled data sets. Each boxplot describes the data from all the adhesions with significant linear fits (p-value below 0.05). (PNG) Figure S10 Reducing the time between each frame only has mild effects on the assembly and disassembly rates in the S178A cells. The label ‘All’ indicates that none of the images were excluded to estimate the rates, while ‘Sampled’ indicates that every other image from each experiment was discarded. To compensate for the shortened experimental time, the minimum number of points needed to determine an assembly or disassembly rate was reduced to 5 for the sampled data sets. Each boxplot describes the data from all the adhesions with significant linear fits (p-value below 0.05). (PNG) Figure S11 Evaluation of the analysis system’s ability to extract quantitative properties from simulated station- ary focal adhesions. (A) The last frame of the stationary simulation, with each adhesion outlined in a color depending on PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e22025