Chamber inserts

SAOS2 and MG63 cells were treated with BAMBI (5 mg/ml) for 24 h at 37°C and used to analyze the cell invasion and migration. SAOS2 and MG63 cells were placed in a 24-well culture plate with chamber inserts (BD Biosciences). For migration assays, 5×10⁴/well SAOS2 and MG63 cells in RPMI-1640 medium were placed into the upper chamber with the non-coated membrane at 37°C for 24 h. For invasion assays, the cells (5×10⁴/well) were placed into the upper chamber with the Matrigel-coated membrane. In the invasion assay, cells were treated with BAMBI (5 mg/ml) for 24 h and subjected to the tops of BD BioCoat Invasion Chambers (BD Biosciences), according to the manufacturer's protocols. The medium and serum in the lower chamber was DMEM plus 20% FBS. The number of tumor cells that invaded and migrated through the membrane were by stained with 0.5% crystal violet at 37°C for 30 min and counted at least three randomly selected fields per membrane under a light microscope in five random visual fields (magnification, ×200).

MCF-7 and BT474 cells were treated with everolimus (5 mg/ml) and cultured for 48 h. Migration and invasion of MCF-7 and BT474 cells was conducted in a 6-well culture plate with chamber inserts (BD Biosciences, San Jose, CA, USA). For migration assays, 1×10⁴/well concentration of the MCF-7 and BT474 cells were placed into the upper chamber. For invasion assays, MCF-7 and BT474 cells (1×10⁴/well) were placed into the upper chamber with the Matrigel-coated membrane. Migration and invasion of MCF-7 and BT474 cells were counted in at least three randomly stain-field microscope every membrane.

HepG2 cells were treated with TIPE-2 (2.0 mg/ml) for 24 h; untreated cells were used as a control. Migration and invasion assays of HepG2 cells were conducted in 6-well culture plates with chamber inserts (BD Biosciences, San Diego, CA, USA). For migration assays, 1×10⁶/well HepG2 cells were placed into the upper chamber with DMEM medium. The lower chamber contained DMEM medium with 0.1% FBS. For invasion assays, cells (1×10⁶/well) were placed into the upper chamber with a Matrigel-coated insert. After 24 h culture at 37°C, migratory and invasive cells were fixed with 1.4% paraformaldehyde for 30 min at 37°C and were stained for 30 min in 0.1% crystal violet solution in PBS. Migration and invasion of HepG2 cells was counted in at least three randomly selected fields per membrane using a light microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan).

A549, pMTA2-, siMTA2-transfected, AbMTA2-treated or PBS-treated cells were seeded into the upper chamber of each insert. Subsequently, 500 µl DMEM containing 10% FBS was added to a 24-well plate for 24 h. Migration and invasion analysis of cells was conducted in a 24-well culture plate with chamber inserts (BD Biosciences). For the migration assays, 1×10⁴ cells/well were placed into the upper chamber with a non-coated membrane. For the invasion assays, cells (5×10⁴ cells/well) were placed into the upper chamber with a Matrigel-coated membrane. All procedures were performed according to the manufacturer's instructions. The cells were fixed and stained for 30 min in a 0.1% crystal violet solution in PBS. The tumor cell invasion and migration was counted in at least three random fields/membrane, by light microscopy (Olympus Corporation, Tokyo, Japan) at ×40 magnification.

Ishikawa cells were treated with cisplatin (5.0 mg/ml) for 24 h and non-treated cells were used as control. Migration and invasion assays of Ishikawa cells were conducted in a 6-well culture plate with chamber inserts (BD Biosciences Franklin Lakes, NJ, USA). For migration assays, Ishikawa cells (1×10⁴/well) were placed into the upper chamber with the non-coated membrane (Transwell inserts; BD Biosciences) and DMEM supplemented with 5% PBS was plated in the lower chamber for 48 h at 37°C. For invasion assays, the cells (1×10⁴/well) were placed into the upper chamber with the Matrigel inserts membrane for 48 h at 37°C. For migration and invasion assays, Ishikawa cells were counted in at least three randomly stained-fields using a light microscope (Olympus Corporation, Tokyo, Japan) at magnification, ×400.

Hepatic carcinoma cells were cultured in DMEM for 48 h at 37°C. Cells were suspended at a density of 1×10⁵ in 500 µl serum-free DMEM. Hepatic carcinoma cells were then plated in the upper chambers of a chamber inserts (BD Biosciences, San Jose, CA, USA) with only DMEM and DMEM with 5% FBS in the lower chambers according to the manufacturer's protocol. In addition, hepatic carcinoma cells (1×10⁶) were incubated with isoflurane (2 mg/kg) or PBS (2 mg/kg) for 72 h at 37°C in a Matrigel-coated membrane (BD Biosciences). The cells were fixed and stained for 30 min in a 0.1% crystal violet solution in PBS. The tumor cell invasion and migration was counted in at least three random fields/membrane, by light microscopy (Olympus Corporation, Tokyo, Japan) at magnification, ×40.