Activation of PKR Inhibits Cell Migration 10 ng/ml) for 36 h and cell migration was determined by wound healing migration assay as described under the Materials and Methods. E: MDA- MB231 cells were exposed to PIC (0, 5, 10, 50 or 100 ng/ml) with/without serum for 24 and 36 h. The cell viability was determined with MTT assay. The number of viable cells after PIC treatment was presented relative to untreated controls. F: MDA-MB231 cells were treated with PIC (0 or 10 ng/ml) for indicated time courses. Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR and eIF2a. The expression of GAPDH served as a loading control. G: The relative levels of pPKR and pelF2a were quantified as described under the Materials and Methods and normalized to the expression of PKR and elF2a, respectively. Each datum point was the mean 6 SEM of three independent experiments. * denotes a statistically significant difference from untreated controls (p,0.05). doi:10.1371/journal.pone.0047721.g001 Figure 2. Effect of PKR inhibitor on PIC-induced inhibition of cell migation. A: MDA-MB231 cells were pretreated with a selective PKR inhibitor (PKR-I, 500 nM) for 24 hours followed by PIC (0 or 10 ng/ml) exposure for 6 hours. Cell lysates were collected for immunoblotting analysis of the phosphorylation/expression of PKR, eIF2a, p38 MAPK and MK2. The expression of actin served as a loading control. B: 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. * denotes a statistically significant difference from untreated groups. # denotes a significant difference from PIC-treated groups. C: MDA-MB231 cells were pretreated with PKR-I (500 nM) for 24 h then placed into the upper compartments of 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. The experiment was replicated three times. * denotes a statistically significant difference from non-PIC-treated groups. # denotes a significant difference from PIC-treated groups. & denotes a significant difference from the untreated group. doi:10.1371/journal.pone.0047721.g002 PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47721