Fig. 7. Actomyosin contractility is not sufficient to activate SRF. (A) Effect of Blebbistatin and Y-27632 on induction of SRF by Ca²⁺ withdrawal. MDCK cells were transfected and treated as before. Blebbistatin (100 µM, 2.5 hours pretreatment) and Y-27632 (20 µM, 30 minutes pretreatment) was added prior to medium exchange and maintained throughout the assay. (B) Rac1 and RhoA activity following Blebbistatin pretreatment. Pull-down assays were performed as in Fig. 5 for Rac1 or RhoA, either 5 or 2 minutes after medium exchange, respectively. (C) Blebbistatin effect on the actin cytoskeleton. Confluent monolayers were pretreated for 2.5 hours with Blebbistatin prior to medium exchange for 7 hours, as in A. Cells were fixed and stained for E-cadherin and F-actin (phalloidin). Shown are micrographs from cells maintained in normal (left) or low (right) Ca²⁺. (D) No effect of constitutively active ROCK4 or the myosin-phosphatase inhibitor calyculin A on SRF activity. Calyculin A was added 30 minutes before medium exchange and maintained throughout the assay. (E) Influences of ROCK4 and calyculin A on the actin cytoskeleton. Cells were transfected or treated as before and stained for myc (ROCK4) and F-actin (phalloidin). Mock, medium exchange with 1.8 mM Ca²⁺; –Ca²⁺, medium exchange with 0.02 mM Ca²⁺; CalA, treated with calyculin A (5 nM). Error bars indicate s.e.m. (n=3). Bars, 50 µm.