Matrigel coated transwell cell culture inserts

For the cell invasion assay, 1x10 4 cells were seeded on Matrigel-coated Transwell cell-culture inserts (Invitrogen Life Technologies, Carlsbad, CA, uSA) with RPMI-1640 medium with 2% FBS. The bottom chamber was filled with 600 µl RPMI-1640 medium with 10% FBS. After 48 h of incubation, cells on the lower surface of the membrane were stained with crystal violet. The number of cells was counted in three random fields under a light microscope.
Animal experiment. Female BALB/c mice at 6 weeks of age were obtained from the National Biological Industry Base, Laboratory Animal Center of Chongqing Medical university. The mice were randomly divided into four groups (five mice per group) and were maintained under pathogen-free conditions. The mice were intravenously injected with MKN-45 cells, and the mice were observed using an imaging system at one and two weeks after inoculation. The mice were then dissected four weeks after inoculation to observe MKN-45 cell metastasis.
Statistical analysis. All experimental data are presented as the means ± SD, and single comparisons between two groups were evaluated by Student's t-test using SPSS 20.0. Associations between the expression levels of HMGA2 and TWIST1 were analyzed by the Pearson correlation coefficient. P<0.05 was considered statistically significant.