Extended Data Fig. 1: AFM-based single cell force spectroscopy (SCFS) to study the effect of PAR-activation on early cell adhesion.

a, Illustration of AFM-based SCFS. A single fibroblast is attached to a tipless concanavalin A-coated cantilever. Subsequently, the cantilever-bound fibroblast is brought into contact with an extracellular matrix (ECM) protein-coated support. After a predefined contact time allowing the fibroblast to establish adhesion to the ECM substrate, the cantilever is retracted to record the force needed to detach the fibroblast from the support in a so-called force-distance curve. b, Exemplary force-distance curves recorded after contact times ranging from 5 to 240 s show distinct features: The retraction force-distance curve (black) records the adhesion force of the fibroblast, which represents the highest force required to separate the fibroblast from the ECM substrate. In total we have recorded 3’500 force-distance curves. c, Exemplary force-distance curves detecting the (un-)binding of single integrins upon separating a fibroblast from a fibronectin fragment (FNIII7-10)-coated support. To record single integrin (un-)binding events with SCFS, fibroblasts are approached to the FNIII7-10-substrate (red curve) until reaching a contact force of ~ 200 pN for a contact time of ~ 150 ms. After this, cantilever and fibroblast are retracted (black/green curve). The green force-distance curve shows a single adhesion event while the black curve shows no adhesion event. In total, we have recorded 12’203 force-distance curves in binding probability assays. d, e, Influence of procedure detaching the fibroblast from cell culture flasks on the adhesion measurement by SCFS. Parental fibroblasts were detached either with (d) 0.25 % (w/v) trypsin or (e) 12 mM EDTA from culture flasks and then attached to the AFM cantilever for SCFS. Dots represent adhesion forces of single fibroblasts, < n(cells) > the number of fibroblasts tested, and black bars median values. Statistical significances (P-values) were determined using two-tailed Mann-Whitney tests and are given if P < 0.05. In the first 30 min after trypsin-induced detachment from culture flasks the adhesion of fibroblasts to FNIII7-10-coated supports increased significantly. 30–60 min after detachment the fibroblasts fully recovered. Adhesion to FNIII7-10ΔRGD or collagen type I was not affected by trypsin detachment. The contact time of fibroblast and ECM-coated supports was 60 s. Consequently, for all SCFS experiments in this work the fibroblasts were allowed to recover from trypsin-induced detachment from culture flasks for at least 30 min, before being attached to the cantilever. f, Experimental assay used for SCFS. Fibroblasts detached from culture flasks using 12 mM EDTA were allowed to recover for 30 min in SCFS media prior to peptide exposure and SCFS (Methods).