Supplementary Figure 10: Essentiality of Rac1 activation in collective migration.
From: A molecular mechanotransduction pathway regulates collective migration of epithelial cells
(a) Phase contrast images of wound closing by control (Left panels), Rac1 inhibitor (NSC 23766)-treated (Middle panels), Rac1-depleted (Right panels) MDCK cells at different time points after confinement removal. Both NSC 23766-treated and Rac1-depleted cells showed negligible migration activity within the investigated time frame. Scale bars, 200 μm. (b) Western blot confirming Rac1-depletion in MDCK cells with Rac1-specific siRNA. (c) Percentage of cells with cryptic lamellipodium, 2 h after removal of confinement, with first 20 layers of cells behind the leading edge. (n = 5 independent experiments; P = 4.92 × 10−8; Wilcoxon rank-sum test). In box plots central mark is the median, and the edges of the box are the 1st and 3rd quartiles. Whiskers extend to the most extreme non-outlier data points. (d) The mutant, M-EzABD, inhibits Rac1 activation even in migration promoting condition. MDCK cells, transfected as indicated, were grown to confluency for 18 h and then were allowed to migrate for 3 h. Western blots showing GTP bound Rac1 (active Rac1) and total Rac1. (e) Bars represent relative Rac1 activation results from densitometric analysis. (mean ± s.e.m; n = 3 independent experiments; P = 2.61 × 10−10; Wilcoxon rank-sum test). Statistics source data are given in Supplementary Table 3. (f) Localization of endogenous Merlin in Jasplakinolide-treated stationary and migrating cells. Even though enhanced actin binding with the M-EzABD construct prevents relocalization, stabilized cortical actin cytoskeleton does not prevent Merlin relocalization. Scale bar, 100 μm. ∗∗∗P < 0.001 (Wilcoxon rank-sum test). Panels (a, b, d, f) show the representative image of 3 independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.