Fig. 5. SRF activation by dissociating epithelial junctions is dependent on Rac, but not on RhoA or Cdc42 activation. (A) Activity of endogenous RhoA, Rac1 and Cdc42 upon Ca²⁺ withdrawal. MDCK cells were seeded to form a confluent monolayer. 36 hours later, the medium was exchanged. After the indicated times, cells were lysed and GST-Rhotekin or GST-PAK-CRIB pull-down assays were employed. Uncoupled beads (control) or lysates incubated with GTPS served as negative and positive controls, respectively. Precipitated proteins (upper panels) and total lysates (lower panels) were blotted using antibodies against RhoA, Rac1 or Cdc42. Pairwise densitometric analysis of the fold induction is shown below each panel. (B) Effect of the inhibition of Rac1 or RhoA on SRF. MDCK cells were transfected and treated as in Fig. 1A. Prior to medium exchange, cells were preincubated with equipotent concentrations of TcdBF (0.1, 0.25 or 0.75 µg/ml) or TcdB (0.3, 1 or 3 ng/ml) for 4 hours, and with Tat-C3 (0.3, 1 or 3 µM) for 15 hours. Inhibitor treatment was maintained throughout the assay. Shown is the mean luciferase activity normalised to protein. (C) Specificity of TcdBF, TcdB and Tat-C3 on Rac1 and RhoA. When indicated, cells were preincubated with TcdBF (0.25 µg/ml, 4 hours), TcdB (1 ng/ml, 4 hours) or Tat-C3 (1 µM, 15 hours). Activity of Rac1 and RhoA was measured as in A, either 5 or 2 minutes after medium exchange, respectively. (D) E-cadherin localisation of cells treated with TcdBF, Tat-C3, TcdB or vehicle. Confocal immunofluorescence micrographs of MDCK cells treated for 11 hours (TcdBF, TcdB) or 22 hours (Tat-C3) in normal medium, as before. Cells were fixed and stained for E-cadherin. (E) Effect of constitutive active Rac1 or RhoA on SRF activity. 2.5 µg RacV12 or RhoQ63L were co-transfected and cells were treated as in Fig. 3A. Shown is the relative SRF luciferase reporter activity normalised to pRL-TK. (F) Effect of the inhibition of RhoA on SRF in NIH3T3 fibroblasts. Cells (35,000 per 1-cm diameter) were transfected with p3DA-Luc (20 ng) and pRL-TK (50 ng). After transfection, cells were maintained in starvation medium (0.5% FCS) for 20 hours before stimulation with 15% FCS. Tat-C3 treatment of NIH3T3 cells was as in B for MDCK cells. Mock, medium exchange with 1.8 mM Ca²⁺; –Ca²⁺, medium exchange with 0.02 mM Ca²⁺. Error bars indicate s.e.m. (n=3). Stars indicate statistical significance at P0.05 according to the Student's unpaired t-test.