Fig. 6. Induction of SRF and endogenous target genes during epithelial dissociation and EMT. (A) Relative SRF luciferase reporter activity in HGF-treated MDCK cells. Cells were transfected and 24 hours later HGF (40 ng/ml) was added for 24 hours prior to analysis of luciferase activity. Phase-contrast images of the cells prior to lysis are shown in B. (C) Effect of TGFβ on SRF activity in EpRas murine mammary epithelial cells. 24 hours after reseeding transfected EpRas cells, EMT was induced by TGFβ (5 ng/ml) for 72 hours. Shown is the relative luciferase activity. Phalloidin-stained cells are shown in D following EMT induction for 7 days. (E) Induction of the endogenous target genes for vinculin (vcl) and smooth muscle -actin (acta2) upon junction disruption. RNA was isolated from confluent EpRas cells 3 hours after medium exchange and analysed by quantitative RT-PCR. Shown is the fold induction of the endogenous SRF target genes in 0.02 mM Ca²⁺, normalised to medium exchange with 1.8 mM Ca²⁺. (F) Effect of inhibiting Rac1 and RhoA on induction of acta2, determined as in E. Where indicated, cells were preincubated with Tat-C3 (1 µM, 15 hours), TcdB (0.3 ng/ml, 4 hours) or TcdBF (0.25 µg/ml, 4 hours). Error bars indicate s.e.m. (n=3). Stars indicate statistical significance at P0.01 according to the Student's unpaired t-test.