However, the signal transduction pathways that mediate migration and invasion induced by OA in breast cancer cells have not been studied in detail. and transferred to nitrocellulose membranes. Next, membranes were blocked using 5% non-fat dried milk in phosphate buffered saline (PBS) pH 7.2/0.1% Tween 20 (wash buffer), and incubated overnight at 4C with primary Ab. The membranes were washed three times with wash buffer and incubated with secondary Ab (horseradish peroxidase-conjugated Abs to rabbit) (1:5000) for 2?h at 22C. After washing, immunoreactive bands were visualized using ECL detection reagent. Autoradiograms were scanned and the labeled bands were quantified using the ImageJ software (https://imagej.nih.gov/ij/). Immunoprecipitation Lysates were clarified by centrifugation at 13,539?for 10?min. Supernatants were transferred to fresh tubes, and proteins were immunoprecipitated overnight at 4C with protein A-agarose linked to a specific Ab against the target protein. Immunoprecipitates were washed three times with RIPA buffer. Scratch-wound assay Cells were grown to confluence in 35?mm culture dishes, starved for 24?h in DMEM and treated for 2?h with 12?M mitomycin C to inhibit proliferation. Next, cultures were scratch-wounded using a sterile 200?L pipette tip, washed twice with DMEM and re-fed with DMEM without or with inhibitors and/or BSA-OA. Progress of cell migration into the wound was photographed at 48?h using an inverted microscope coupled to camera. Each experiment was repeated three times. Invasion assay Invasion assays were performed by the modified Boyden chamber method in 24-well plates containing 12 cell-culture inserts with 8?m pore size (Costar, Corning, Inc). An amount of 50?L BD Matrigel was added into culture inserts and kept overnight at 37C to form a semisolid matrix. Cells were plated at 1??105 cells per insert in serum-free DMEM on the top chamber. The lower chamber contained 600?L DMEM without or with BSA-OA. Chambers were incubated for 48?h at 37C in a 5% CO2 atmosphere, and then cells and VU6005649 Matrigel on the upper surface of membrane were removed with cotton swabs, and cells on the lower surface of membrane were washed and fixed with methanol for 5?min. Number of invaded cells was estimated by staining with 0.1% crystal violet in PBS. Dye was eluted with 300?L 10% acetic acid, and absorbance at 600?nm was measured. Background value was obtained from wells without cells. Determination of 12(S)-HETE MDA-MB-231 cells were treated with 100?M OA or 15?M AA for 30?min, and supernatants were collected. The concentration of 12(S)-HETE was determined by using the 12(S)-HETE ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA), according to the manufacturers guidelines. RNA interference AKT2 expression was silenced in breast cancer cells by using the Silencer siRNA kit from Santa Cruz Biotechnology, according the manufacturers guidelines. One control of scramble siRNAs was included according to the manufacturers guidelines. Silencing of FFAR4 with shRNA Lentiviral shRNA vectors from Santa Cruz Biotechnology targeting human FFAR4 TM4SF2 were utilized for generation of stable knockdown in MDA-MB-231 cells, according the manufacturers VU6005649 guidelines. Transfected cells were selected by their resistance to puromycin (5?g/mL). Immunofluorescence confocal microscopy Cells grown on coverslips were stimulated with OA for various times. After stimulation, cells were fixed with 4% paraformaldehyde in PBS for 20?min, permeabilized with 0.1% Triton X-100 in PBS for 20?min, and blocked for 1?h with 3% BSA. Cells VU6005649 were stained with TRITC-conjugated phalloidin to reveal F-actin and with anti-paxillin Ab for 12?h to reveal focal adhesions, followed by incubation with FITC-labeled anti-mouse secondary Ab for 2?h at room temperature. Cells were viewed using a Leica confocal microscope (Model TCS SP2; Leica Microsystems). Serial optical sections of 0.8?0.9?m thick were taken in both xyz and xzy. To prevent interference from the fluorescent probes, images of the same optical section were taken as separate channels, and they were analyzed by using ImageJ software. Preparation of nuclear extracts Briefly, 1.5??106 cells were lysed with 0.1% nonionic detergent Nonidet P40 in Buffer A (10?mM Tris-HCl, pH 7.4, 10?mM NaCl, 6?mM MgCl2, 10?mM NaF, 1?mM Na3VO4, 1?mM DTT, 1?mM PMSF). Lysates were pelleted at 636?for 15?min and resuspended in Buffer B (20?mM HEPES, pH 7.9, 420?mM NaCl, 20% glycerol 1.5?mM MgCl2, 0.2?mM EDTA, 1?mM Na3VO4, 10?mM VU6005649 NaF, 1?mM DTT, 0.2?mM PMSF). Nuclear extracts were recovered by centrifugation at 17,136?for 15?min at 4C and.