Figure 3 - Rho–ROCK function is required only in stromal fibroblasts for collective SCC invasion.

(a) Matrix remodelling and track generation by control or inhibitor-treated fibroblasts is shown by reflectance (white) and F-actin (red) imaging. The concentration of matrix and appearance of holes around control cells is indicative of matrix remodelling. (b) Representative images of SCC12 cells transfected with GFP–CAAX (green) and HN-CAFs transfected with mRFP–CAAX (red) invading into naïve matrix (grey in reflectance imaging) in the absence or presence of the ROCK inhibitor Y27632. The arrow indicates direction of invasion (volume rendered, approximately 200 200 40 m). (c) Quantification of SCC12-cell invasion in an organotypic assay in the absence of stromal fibroblasts, in the presence of stromal fibroblasts, with Rho function inhibited (Tat-C3 toxin) or with ROCK function inhibited (by siRNA) in either stromal fibroblasts or SCC12 cells (n = 3). The single asterisk indicates P <0.05. (d) Quantification of A431 cell invasion in an organotypic system in the presence of control stromal fibroblasts or those with Rho function inhibited by Tat-C3 toxin (n = 2). (e) Quantification of SCC12 cell invasion in matrix that previously contained stromal fibroblasts is shown on the left. Rho, ROCK or MMP function inhibited by Tat-C3 toxin, Y27632 or GM6001, respectively, in fibroblasts before their removal and addition of SCC cells (n = 2). Quantification of SCC12 cell invasion in matrix that previously contained stromal fibroblasts without any inhibitors. Rho, ROCK or MMP function was inhibited by Tat-C3 toxin, Y27632 or GM6001, respectively, after the fibroblasts had been removed when only SCC cells were present. The double asterisk indicates P <0.01. The error bars indicate s.d. The scale bars represent 10 m in a.