In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes and metastatic properties (promoter. Primer sequences are listed in Supplementary Table S1. Immunofluorescent imaging of F-actin cytoskeletal structure Immunofluorescent imaging was performed according to a reported procedure (34). In brief, treated cells were washed with cold PBS, fixed with 4% formaldehyde for 10 min at 37C, permeabilized with 0.5% Triton X-100 for 5 min at room temperature, and then blocked with 3% BSA overnight at 4C. After washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated phalloidin in the presence of 1% BSA for 1 h at room temperature (for F-actin). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) contained in the Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Confocal images were obtained with an Olympus FV1000 confocal microscope (Olympus Corp., Japan) using the 40 oil immersion lens. In vitro Migration and Invasion Assays Assays were performed buy 197855-65-5 using Falcon? cell culture inserts (8 m pore size) in a 24-well format (BD Biosciences) according to the vendors instructions. In the migration assay, cells (104 cells/well) in 0.5 mL of serum-free medium containing OSU-53 at the indicated concentration were seeded onto the membranes of the upper chambers, which had been inserted into the wells of 24-well plates containing 10% FBS-supplemented medium. After 18 h, the cells were fixed with 100% methanol and stained with 5% Giemsa (Merck, Darmstadt, Germany). Unmigrated cells remaining in the upper chambers were removed by wiping with a damp cotton swab leaving those that had migrated to the underside of the membranes. The membranes were mounted on glass slides, and the numbers of cells in three randomly chosen high power fields were counted. For the invasion assay, cells (105 cells/well) in 0.5 mL of serum-free medium containing OSU-53 at the indicated concentration were seeded onto Matrigel-coated membranes of the upper chambers. The lower chambers contained the same amount of OSU-53 in 10% FBS-supplemented medium. After 24 h, noninvasive cells remaining on the upper surface of the membranes were removed with a cotton swab. Cells on the lower surface of the membrane were fixed in 100% methanol and stained with 0.1% crystal violet for 10 min. The membranes were mounted on glass slides, and the numbers of cells in three randomly chosen high power fields were counted. All experiments were performed three times. Three-dimensional Colony Formation Assay Cells were cultured in growth factorCdepleted three-dimensional Cultrex Basement Membrane Extract (BME) (Trevigen, Gaithersberg, MD), as previously reported (36). In brief, cell culture dishes (24-well plates) were pre-coated with undiluted phenol red-free BME. Cells (104 cells per well) were suspended in 200 L serum-free medium, and then mixed with 100 L of cold BME. The cell suspension was added dropwise onto the BME layer in the pre-coated wells. After the cell-containing layer was set, serum-free medium containing OSU-53 at the indicated concentrations was added over the top. Medium was changed every three days. After culture for 9 and 16 days for PC-3 and MDA-MB-231 cells, respectively, cells were fixed with 4% paraformaldehyde for 20 min, quenched with 0.75% glycine three times, 10 min each, and then examined microscopically for stellate morphology of colonies indicative of invasiveness and migratory Rabbit Polyclonal to TCEAL3/5/6 capacity. In vivo Metastasis Study Orthotopic xenograft tumors were established in female BALB/c mice (BALB/cAnNCr; 5C7 weeks of age; NCI, Frederick, MD) by injecting 4T1 cells (2.5 104 cells/mouse) into the right inguinal mammary fat pad in a buy 197855-65-5 total volume of 0.1 mL of PBS. Mice were randomized to three groups (n = 6), which received the buy 197855-65-5 following treatments 24 h after implantation: (a) OSU-53 at 50 mg/kg; (b) OSU-53 at 100 mg/kg; and (c) vehicle (0.5% methylcellulose/0.1% Tween 80 in water). Treatments were administered once daily by oral gavage. Primary tumor volumes were calculated from weekly caliper measurements (volume = width2 length 0.52). Body weights were measured weekly. At terminal sacrifice, tumors were harvested, snap-frozen in liquid nitrogen, and stored at ?80C until used for biomarkers assessment by Western blotting. Lungs were collected and.