Extended Data Fig. 9: PAR2 activation affects fibroblast spreading and adhesion maturation on the longer term.

a, Integrin adhesion maturation and spreading of a representative fibroblast, as seen by monitoring recruitment of the adaptor protein paxillin to adhesion sites over time. Parental fibroblasts in suspension were exposed for 15 min to 0.1 mM PAR2-activating or control peptide prior to experiments. Subsequently the fibroblasts were seeded into FNIII7-10-coated glass-bottom wells containing 0.1 mM PAR2-activating or control peptide in 100 µl DMEM (without FCS). Timelapse confocal microscopy was used to image (frame time 34 s) paxillin-GFP. Time between images shown here is 2.8 min. Scale bar, 20 µm. b, Classification result of a representative fibroblast, at one time point (40.2 min). After imaging, the spreading of single fibroblasts could be analyzed. For each single fibroblast and in every image (one per 34 s), each pixel was evaluated using the object classification program ilastik. Eight fibroblasts (4 per condition) were used as training set for the classification. We trained the algorithm to recognize background, cytoplasm, nucleus and adhesion site by intensity, edges and texture. The classification was then applied to all fibroblasts (n = 42 and n = 51, for PAR2-activating or control peptide, respectively). Finally, the output for each pixel is the probability of being background, cytoplasm, nucleus or adhesion site (total probability of 1). The analysis and classification procedures were used to determine the fibroblast spreading area, total adhesion size shown in Fig. 5a–c. Scale bar, 20 µm. The experiment was repeated 10 times with at least four cells analyzed per repeat and condition providing the similar results.