To our knowledge, this is the first report demonstrating that Tg737 contributes to hypoxia-induced invasion and migration in HCC

cells. The results of this research indicate that Tg737 may play a role in HCC gene therapy and should be investigated further. Materials and methods Cell line and culture condition HepG2 and MHCC97-H NSC23766 manufacturer cells (maintained in our laboratory, originally obtained from the Cell Bank of Type Culture Collection of the check details Chinese Academy of Sciences), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 IU/ml penicillin, 400 IU/L trypsin, and 100 μg/ml streptomycin and were plated in 75-cm2 flasks and cultured at 37°C with 5% CO2 and 95% humidified air. The medium was changed every 2 days. In all subsequent

related experiments, the HepG2 and MHCC97-H cells were treated with medium supplemented with 1% FBS, unless otherwise noted. For the incubation of cells selleck under hypoxic conditions, the cells were exposed to 1% O2 with 5% CO2 at 37°C for the indicated times. Annexin V/propidium iodide (PI) assay To exclude the possibility of apoptosis-related effects in subsequent experiments, Annexin V/propidium iodide assays were performed. After 18 h of incubation with medium supplemented with 1% FBS medroxyprogesterone under normoxic or hypoxic conditions at 37°C, the cells were harvested, washed in cold phosphate-buffered saline (PBS), incubated for 15 min with fluorescein-conjugated Annexin V and PI and analyzed using flow cytometry. The cells incubated with medium supplemented with 10% FBS under normoxic conditions were also analyzed. Adhesion assay An adhesion assay was performed in 12-well plates as described elsewhere [9]. After 10 h of incubation with medium

supplemented with 1% FBS at 37°C under normoxic or hypoxic conditions, the cells were harvested, resuspended (1 × 105 in 1.5 ml of DMEM supplemented with 1% FBS), plated onto collagen surfaces, and allowed to adhere for 2 h, consistent with the previous conditions (normoxia or hypoxia). The unbound cells were removed by washing twice with PBS, and the adherent cells were counted under a microscope at 200× magnification from 10 random fields in each well. Each experiment was performed in triplicate. Cell invasion and migration assays Cell migration was assayed using transwells with 8-μm pore filters (Costar, MA, USA). The lower chamber was filled with DMEM supplemented with 10% FBS and 5 μg/ml of fibronectin (Sigma, St. Louis, MO, USA), and 2 × 104 cells in 0.5 ml of media supplemented with 1% FBS were loaded into the upper chamber.