events including cytoskeleton reorganization, membrane ruffling and lamellipodia/filopodia formation [12,13]. In this study, we investi- gated the involvement of PKR in regulation of cell motility/ migration. We demonstrated that the activation of PKR inhibited the motility/migration of breast cancer cells. We further showed that activation of PKR suppressed cofilin activity. Cofilin is an F-actin severing protein that regulates actin depolymerization, a process required for the changes in cell shape and the formation of lamellipodia during migration [14,15]. Cofilin plays an important role in the invasion and metastasis of breast cancer cells [16,17]. We further delineated a signal transduction pathway that mediated PKR regulation of cofilin activity. Materials and Methods Materials Human plasma fibronectin was obtained from Chemicon International (Temecula, CA). Anti-phospho-eIF2a (Ser51) antibody and Lipofectamine 2000 were purchased from Invitrogen Corpora- tion (Carlsbad, CA). Antibodies for phospho-p38 MAPK, phospho- cofilin, cofilin, phospho-LIMK, LIMK, phospho-MAPKAPK2 (MK2) and MK2 were purchased from Cell Signaling Technology Inc. (Beverly, MA). Antibodies for PKR, GAPDH, a-actin, eIF2a and p38a were purchased from Santa Cruz Biotechnology (San Cruz, CA). Anti-phospho-PKR antibody was obtained from Abcam (Cambridge, MA). Phalloidin 488, Alex Fluor-labeled secondary antibodies and Prolong Gold anti-fade reagent were obtained from Invitrogen Molecular Probes (Eugene, OR). MTT assay kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Transwells were obtained from Costar Corp. (Acton, MA). Polyriboinosinic acid [poly I]-polyribocytidylic acid [poly C] (PIC) was purchased from InvivoGen (San Diego, Ca). Inhibitors for PKR, p38 MAPK, ERK and JNK were obtained from Millipore (Billerica, MA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Cell Culture and PIC Treatments MDA-MB231, BT474 and SKBR3 human breast cancer cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). MDA-MB231 breast cancer cells were grown in DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotics-antimycotic (Gibco, Grand island, NY) at 37uC with 5% CO2. MDA-MB231 cells were used for most of the experiments because they have high invasive potential and low PKR expression/activity. BT474 cells were grown in RPMI 1640 medium containing 10% FBS, 1% antibiotics-antimycotic and 10 mg/ml insulin at 37uC with 5% CO2. SKBR3 cells were grown in IMEM medium containing 10% FBS and 1% antibiotics- antimycotic at 37uC with 5% CO2. PIC, a synthetic analog of dsRNA, was used to activate PKR. For PIC treatment, Lipofectamine 2000 was included to facilitate PIC entry into the cells. For controls, cells were treated with Lipofectamine 2000 alone. The concentrations of PIC for this study ranged from 1– 100 ng/ml. For treatment with PKR inhibitor, cells were pretreated with the PKR inhibitor (500 nM) for 24 hours prior to PIC exposure. For treatment with p38 MAPK inhibitor (SB203580), cells were pretreated with SB203580 (5 mM) for 2 hours, then exposed to PIC. PKR siRNA Transfection Transient transfection of PKR siRNA (San Cruz, CA) was performed with Lipofectamine 2000 according the manufacturer’s protocol. Briefly, MB231 cells were treated by control siRNA (con siRNA) or PKR siRNA at a concentration of 200 nM in the Activation of PKR Inhibits Cell Migration presence of Lipofectamine 2000. After 48 hours of incubation, cells were exposed to PIC and cell signaling events or cell migration were assayed. Cell Migration Cell migration was analyzed using a Transwell Migration System (Costar) as described before [18]. Briefly, cells (56104 cells) were placed into the upper chambers (Transwells with 8.0 mm pore size) in serum free medium in the presence or absence of PIC. Culture medium containing 10% FBS was added into the lower compartment of the chambers and served as a chemoattractant for the cells. The chambers were cultured at 37uC in 5% CO2 overnight. The migrated cells were fixed in 3.7% paraformalde- hyde and stained with 0.5% crystal violet in 2% ethanol. Membranes were washed and the dye was eluted with 10% acetic acid. Absorbance was measured at 595 nm using a microtiter platereader (Beckman coulter). PIC or chemical inhibitors were only added to the upper chambers. Wound Healing Migration Assay The wound healing migration assay was performed as described previously [18]. MDA-MB231 cells were grown on 35 mm dishes to 100% confluence and then scratched to form a wound using sterile pipette tips. The cells were then treated with PIC (0, 1, 5 or 10 ng/ml) for 36 hours. The images were recorded using a Zeiss Axiovert 40C photomicroscope. MTT Assay The MTT assay was employed to determine the number of viable cells in culture. Briefly, the cells were plated into 96-well plates and exposed to PIC for 24 hours. After the treatment, 10 ml of MTT reagent was added into each well and the plates were incubated at 37uC for 4 hours. The cultures were solubilized and spectrophotometric absorbance was measured at 595 nm using a microtiter platereader. Analysis of Cell Morphology Cell adhesion to fibronectin was analyzed as described previously [19,20]. Briefly, cells were exposed to PIC for 12 hours. After exposure, cells (56104/well) were seeded on fibronectin (10 mg/ml)-precoated plates, allowing attachment for 3 hours at 37uC with 5% CO2. Non-adherent cells were removed by washing with PBS. The attached cells were fixed with 3.7% paraformaldehyde for 10 min, then washed 3 times in PBS. The phase-contrast images were captured using a Zeiss Axiovert 40C photomicroscope. Cells with protruded leading areas (lamellipo- dia) were counted in ten randomly selected fields in each treatment group. Cell spreading areas were measured using Motic Images Plus 2.0 ML software (Motic, Canada). Immunofluorescence Microscopy The procedure for immunofluorescence microscopy has been previously described [21]. Briefly, after treatments, cells were fixed with 3.7% paraformaldehyde for 10 min, washed 3 times in PBS and permeabilized with 0.5% Triton X-100 for 5 min. Cells were blocked with 5% BSA and incubated with primary antibodies for 1 hour. The concentrations of primary antibodies were: anti-cofilin, 1:100; anti-phospho-cofilin, 1:100; anti-phospho-MAPKAPK2, 1:100; and phalloidin, 1:200. After the incubation, cells were washed and treated with Alexa Fluor-labeled secondary antibodies and rinsed several times with PBS. Coverslips were mounted with Prolong Gold anti-fade reagent and immunofluorescence images were examined with an Olympus 1X81 inverted fluorescent PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47721