We previously showed that Fyn, a member of Src family tyrosine kinase (Src-TK), was involved in the activation of Rho-kinase (ROK) to develop Ca2+ sensitization of vascular smooth muscle contraction induced by sphingosylphosphorylcholine (SPC). Recently we found that SPC can stimulate the formation of stress fibers and filopodia-like protrusions in NIH3T3 fibroblasts through Src-TK/ROK-dependent and independent pathway, respectively. Then, we further investigated the role of Fyn in the SPC-induced formation of stress fibers and filopodia-like protrusions with RNA interference method. Three different Fyn siRNAs were designed and transfected into NIH3T3 cells using Lipofectamine 2000. As control, a non-silencing siRNA and a positive MAPK1 control siRNA were transfected in parallel. The non-silencing AF 488-labeled siRNA was used to monitor transfection efficiency. Our results showed that transfection efficiency was high above 90% and the down-regulation of Fyn expression was confirmed in western blot with concentration-dependency and incubation time-dependency. SPC-induced stress fiber formation was partially inhibited by Fyn siRNAs, but not by other control siRNAs. The formation of filopodia-like protrusions induced by SPC was not affected by Fyn siRNAs. These findings suggest that Fyn plays a role in SPC-induced stress fiber formation, but not in the formation of filopodia-like protrusions in NIH3T3 cells. [J Physiol Sci. 2006;56 Suppl:S70]
