Ix2 microscope

For the migration assay, Renca cells were plated in six-well plates at a density of 3 × 10⁵ and incubated with medium alone or serial dilutions of lycorine hydrochloride (0, 1, 2.5, 5, and 10 μM) for 24 h. Renca cells were resuspended in serum-free medium and seeded into trans-well chambers with 8-μm diameter pore size polycarbonate membranes (Corning, NY, USA). Medium containing 30% fetal bovine serum was used in the lower chamber as an attractant. For the invasion assay, trans-well chambers were coated with Corning Matrigel Matrix (Corning, NY, USA). After 12 h, cells on the upper chamber surface were removed using cotton swabs. The migrated or invaded cells in the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The total numbers of cells were captured and analyzed from 10 different fields with the Olympus IX2 microscope.

Renca cell migratory ability was evaluated via the scratch motility assay. Cells were seeded in 6-well plates (Costar, Corning, NY, USA) at a density of 3 × 10⁵ for 24 h. A vertical wound was made with a sterile pipette tip, followed by washing with PBS two times, and then incubated with medium alone or serial dilutions of lycorine hydrochloride (0, 2.5, 5, and 10 μM) for 24 h at 37°C and 5% CO₂. Images of cell migration onto these scratched zones were captured by the Olympus IX2 microscope (Olympus, TOKYO, JAPAN). The scratch width was calculated using the Image Pro Plus 6.0 software. Migration rate was calculated as (%) = [1 −*W (24 h)]/*W (0 h), where *W (0 h) and *W (24 h) indicate the width of the wound at 0 h and 24 h, respectively.

For the migration assay, cells were plated in six-well plates at a density of 3 × 10⁵ and incubated in DMEM-0% FBS (serum-free medium) in the presence of BsAb for 8 h and JNJ for 2 h, then were treated with HGF (100 ng/ml). Cells were resuspended in serum-free medium and seeded into transwell chambers with 8-μm diameter pore size polycarbonate membranes (Corning, NY, U.S.A.). Medium containing 30% FBS was used in the lower chamber as an attractant. After 12 h, cells on the upper chamber surface were removed using cotton swabs. The migrated or invaded cells in the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% Crystal Violet. The total numbers of cells were captured and analyzed from ten different fields with the Olympus IX2 microscope.