Activation of PKR Inhibits Cell Migration Figure 6. Effect of PKR siRNA on PIC-regulated cell migation. MDA-MB231 cells were transfected with control siRNA or PKR siRNA for 48 h. A: Following transfection, cell lysates were collected and the expression PKR was determined by immunoblotting. B: Following transfection, MDA- MB231 cells were placed into the upper compartments of the migration chambers in the presence of PIC (0 or 10 ng/ml). The number of MDA-MB231 cells that migrated through the transwells was measured as described under the Materials and Methods. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated groups. C: Following transfection, MDA-MB231 cells were exposed to PIC (0 or 10 ng/ml) for 6 hours. Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, eIF2a, p38 MAPK and MK2. The expression of tubulin served as a loading control. D: The relative levels of pPKR, pelF2a, pp38 and pMK2 were quantified as described under the Materials and Methods and normalized to the expression of PKR, elF2a, p38 MAPK and MK2, respectively. The experiment was replicated three times. * denotes a statistically significant difference from controls. # denotes a significant difference from PIC-treated groups. doi:10.1371/journal.pone.0047721.g006 Activation of PKR Inhibits Cofilin through p38 MAPK/ MK2/LIMK Pathways We next sought to determine the mechanisms for PIC-induced actin cytoskeleton reorganization and decreases in lamellipodia formation. Cofilin is an F-actin severing protein and plays an important role in the reorganization of the actin cytoskeleton. Cofilin activity is negatively regulated by phosphorylation at serine 3; phosphorylation of cofilin renders it unable to depolymerize F- actin, thereby stabilizing the cytoskeleton [22]. We examined the effect of PIC on cofilin activity. PIC increased the phosphorylation of cofilin (Ser3) (Figs. 4A and B). PIC-induced cofilin phosphor- ylation was confirmed by immunofluorescent staining (Fig. 5C). In the control group, active cofilin (non-phosphorylated form) was distributed at the leading edges of cells, i.e. lamellipodia (Fig. 4C). In the PIC-treated group, however, no active cofilin was observed at the leading edges. Since the localization of active cofilin at the PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47721