Activation of PKR Inhibits Cell Migration Figure 3. Activation of PKR impairs lamellipodia formation. MDA-MB231 cells were treated with PIC (0, 1, 5 or 10 ng/ml) for 12 hours, and then equal amounts of cells were seeded on fibronectin-coated culture wells, allowing attachment for 3 hours. A: After attaching, the phase-contrast images were captured using a Zeiss Axiovert 40C photomicroscope. The images of cells treated with PIC (0 or 10 ng/ml) are presented. Scale bar = 50 mm. B: Cells with extended leading areas (lamellipodia) were counted in ten randomly selected fields in each treatment group. The percentage of cells with lamellipodia was determined. C: MDA-MB231 cells were treated with PIC (0 or 10 ng/ml) for 12 hours. The distribution of the actin cytoskeleton was detected by fluorescent staining (Alexa Fluor 488 Phalloidin) as described under the Materials and Methods. The arrow indicates the lamellipodia (the leading edge). Scale bar = 10 mm. D: MDA-MB231 cells were treated with PIC (0, 1, 5 or 10 ng/ml) for 12 hours, and then equal amounts of cells were seeded on fibronectin-coated culture wells, allowing attachment for 3 hours. Cell spreading areas were measured randomly for at least 25 cells for each treatment group. The experiment was replicated three times. Each datum point was the mean 6 SEM of three independent experiments. * denotes a significant difference from untreated controls. # denotes a significant difference from PIC (1 ng/ml)-treated groups. 1 denotes a significant difference from PIC (5 ng/ml)-treated groups (p,0.05). doi:10.1371/journal.pone.0047721.g003 microscope with the same exposure time, detector gain and amplifier offset. Immunoblotting The procedure for immunoblotting has been previously described [19]. Briefly, after PIC treatments, cells were lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris, 1% NP-40, 0.25% sodium deoxycholate, 1 mM sodium vanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF), 5 mg/ml of aprotinin, and 2 mg/ml of leupeptin). Samples were separated by centrifu- gation at 10,000 rpm for 10 min at 4uC. Proteins were resolved in SDS-PAGE and the separated proteins were transferred to nitrocellulose membranes. The membranes were probed with indicated primary antibodies, followed by the appropriate secondary antibodies and developed by enhanced chemilumines- cence. The images of immunoblots were documented using Gel Logic 2200 Pro (Carestream Health, Rochester, NY). The intensity of specific proteins was quantified using Carestream Molecular Image Software. Time-lapse Microscopy The rate of cell membrane ruffling was determined by time- lapse microscopy. After treated with PIC for 12 hours in the presence or absence of inhibitors, cells were trypsinized and seeded on fibronectin precoated glass-bottom dishes in medium contain- ing 20 mM HEPES. Cells were allowed to attach for 3 hours at 37uC with 5% CO2. Cells were maintained at 37uC and recorded by a phase-contrast time-lapse video program using an Olympus 1X81 inverted fluorescent microscope with a 60X oil immersion lens. The recording was performed at 10-second intervals for 10 min. Kymographs were generated from time-lapse video images using ImageJ software (NIH) as previously described [14]. Five or eight 1-pixel-thick lines were drawn across the cell leading edges and the pixel intensities along each line were combined to create the kymographs. To determine the dynamic frequency of cell membrane ruffling, the perceived changes of waves in 10 min on kymographs were counted manually and presented relative to the control groups. Statistics Differences among treatment groups were analyzed using analysis of variance (ANOVA). Differences in which p was less than 0.05 were considered statistically significant. In cases where significant differences were detected, specific post-hoc comparisons between treatment groups were examined with Student-Newman-Keuls tests. Results Activation of PKR Inhibits the Motility of Breast Cancer Cells We first examined the effect of PIC on the motility/migration of breast cancer cells. As shown in Fig. 1A, PIC suppressed the migration of MDA-MB231 cells which was determined by transwell assay in a concentration-dependent manner. Even at 1 ng/ml, PIC significantly inhibited the migration of MDA- MB231 cells. Similar effects of PIC were observed in other aggressive breast cancer cells (BT474 and SKBR3) (Fig. 1B). We examined the expression/phosphorylation of PKR in different breast cancer cell lines. As shown in Fig. 1C, the levels of PKR expression/phosphorylation were much lower in more aggressive breast cancer cell lines (MDA-MB231, BT474 and SKBR3) than in the less aggressive line (MCF-7). PIC-mediated inhibition of cell migration was confirmed by the wound healing assay; PIC treatments impaired MDA-MB231 cell movement in a concen- tration-dependent manner (Fig. 1D). The inhibition of cell motility by PIC was not due to a decrease in cell viability because PIC up to 10 ng/ml did not alter cell viability within 36 hours of exposure (Fig. 1E). PIC-induced activation of PKR was verified by an increase in the phosphorylation of PKR (Thr446) and eIF2a (Ser51), a substrate of PKR in MDA-MB231 cells (Figs. 1F and G). To determine whether PIC inhibition of cell migration was indeed mediated by PKR, we used a PKR inhibitor (PKR-I) to block PKR activity. As shown in Fig. 2A, PKR-I inhibited PIC- mediated phosphorylation of PKR and eIF2a. PKR-I blocked PIC-induced phosphorylation of p38 MAPK and pMK2, indicat- ing that p38 MAPK/pMK2 was downstream of PKR (Figs. 2A and B). More importantly, PKR-I alleviated PIC-induced inhibi- tion of cell migration (Fig. 2B). PKR-I also reduced PIC-mediated formation of lamellipodia (data not shown). These data indicated that activation of PKR suppressed the motility of breast cancer cells. Activation of PKR Impairs Lamellipodia Formation The formation of lamellipodia at the leading edge of cells is characteristic of cells in motion. Since PIC inhibited cell motility, we sought to test whether PIC affected the formation of lamellipodia. MDA-MB231 cells were pretreated with PIC for 12 hours, allowed to attach to fibronectin for 3 hours and examined under the microscope. In the control group, the majority of cells formed a protrusive lamella (lamellipodia) (Figs. 3A and B); however, in the PIC-treated group, the number of cells with lamellipodia was significantly reduced. Since dynamic polymerization/depolymerization of the actin cytoskeleton is required to generate the force to form lamellipodia, we sought to determine whether PIC altered the organization of the actin cytoskeleton. In the control group, the actin cytoskeleton was cumulated at the lamellipodia and formed a polarized actin-rich leading edge (Fig. 3C). In the PIC-treated group, few cells displayed this feature. In addition, PIC also decreased cell spreading areas (Fig. 3D). Thus, PIC inhibited the reorganization of the actin cytoskeleton and impaired lamellipodia formation; these alterations may underlie PIC-induced inhibition of cell motility. PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47721