Traction force microscopy was performed as previously described (Sabass et al., 2008 (link); Oakes et al., 2012 (link)). In brief, PA gels with a Young’s modulus of 8.4 kPa were polymerized with 80-nm fluorescent microspheres (Invitrogen) on prepared glass coverslips and covalently conjugated with collagen 1 (BD) using sulphosanpah (Thermo Fisher Scientific). Cells were plated on gels and allowed to spread overnight. As cells readily formed pairs on PA gels, cell pairs were analyzed rather than single cells. Images of microspheres were captured before and after cell removal with a 0.5% SDS solution, and image pairs were registered and analyzed by PIV as in the previous section. Traction stresses were calculated from the displacement field by Fourier transform traction cytometry using zeroth-order regularization.