Supplementary Figure 5: Quantitative imaging of siRNA- or Arp2/3 inhibitor-treated cells.
(a) Representative images illustrating segmentation of U2OS cells based on DAPI (nuclei) and F-actin staining and subsequent segmentation of mCherry-vinculin and integrin β5-2GFP fluorescence (shown for an individual cell; cropped). Images representative of n = 2 biologically independent experiments. Scale bars 50 µm (10 µm in cropped images). (b) Representative images of U2OS cells stained with DAPI (nuclei) and phalloidin, and immunolabelled for integrin β5 and vinculin following treatment with Arp2/3 inhibitor (CK-666) or inactive analogue control (CK-689). Scale bars = 50 µm. (c) Boxplots summarizing single cell quantification (from images as shown in B; n = 1019 cells analysed, averaging 509 + /- 23 (stdev) per condition) of reticular to focal adhesion integrin β5 ratios following treatment with Arp2/3 inhibitor (CK-666) or inactive analogue control (CK-689). Boxplot centre and box edges indicate median and 25th or 75th percentiles, respectively, while whiskers indicate the median + /- 1.5*IQR (inter-quartile range) or the most extreme observations within these limits. Boxplot notches approximate 95% confidence intervals (CI; see methods for details). P-values reflect two-sided unpaired Mann Whitney U testing. Images and data in B-C derived from 3 biologically independent experiments. Source data for C is available in Supplementary Table 1
