Fig. 6. Effects of reduced PLD and Rho kinase activities on S1P-induced stress fibres and focal adhesions. C2C12 myoblasts transfected with DN-PLD were stimulated with 1 µM S1P for 30 minutes (A) or pretreated with 50 µM Y-27632 prior to stimulation (B). The cells were then fixed and double stained with TRITC-phalloidin (red) and anti-vinculin (green). Of interest, the combined treatment is able to abrogate completely stress fibre and focal adhesion formation in response to S1P. (C,D) Time-lapse analysis of C2C12 myoblasts expressing GFP-actins transfected with DN-PLD by two-photon excitation fluorescence microscopy. Images were taken 1 minute (C) and 30 minutes (D) after the application of 1 µM S1P. A complete filament dispersion is visible after 30 minutes from stimulation. The images are representative of at least three independent experiments with similar results.