Fig. 3. Role of S1PR in the cytoskeletal response of S1P. Intracellular S1P stimulation of native C2C12 myoblasts: cells were microinjected with a mixture of 500 µM S1P and 5 mg/ml PI (A). Extracellular stimulation of native C2C12 myoblasts with 1 µM S1P: cells were microinjected with PI solely as a tracking agent (B). Extracellular stimulation of native C2C12 myoblasts after pretreatment with PTx (C). In all the experimental conditions, the cells were fixed after 30 minutes of stimulation and F-actin organization was visualized by Alexa488-phalloidin. Note that, in contrast to the extracellular stimulation (B), the intracellular delivery of S1P (A) is unable to modify the assembly of stress fibres. However, the inhibition of Gi-coupled receptor pathway by PTx reduces the formation of F-actin filaments by S1P. The images are representative of at least three independent experiments with similar results.