The effects of myosin-II contractility on collective migration on soft and stiff substrates. (A) Maximum intensity projection of z-stack images of collectively migrating cells on soft and stiff substrates immunostained with pMLC (green), phalloidin as actin marker (red), and DAPI as the nuclei marker (blue). Bar, 50 µm. (B) Spatial variation of pMLC fluorescent intensity normalized by cell numbers. Error bars show SEM. (C) Focal adhesion lengths on stiff and soft substrates. Blue bars within box plots indicate means and SEM; data points outside the whiskers are outliers. (D) Effects of 25 µM blebbistatin treatment on the mean speed of individual cells at 12 h after wounding on soft and stiff substrates. (E and F) Effects of 25 µM blebbistatin on mean normalized cell pair separation distance (E; after 5 h of observation starting 8 h after wounding, see Fig. 2) and velocity correlation at 12 h after wounding (F) on soft and stiff substrates for cells 160 µm from the wound edge. N = number of experiments; n = total number of cells measured from N experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Nonoverlapping error bars in D–F (95% SEM) are statistically significant with P < 0.05.