Cell invasion and migration

To identify cell invasion ability, we used a high throughput screening multi‐well insert 24‐well two‐chamber plate (BD Biosciences, San Jose, CA), with an 8‐μm (pore size) polycarbonate filter between chambers. 2.5 × 10⁴ cells of nA8‐Rap1A cDNA, HEY‐Rap1A sh1, SKOV3‐Rap1A sh2, and their corresponding controls were placed into the upper chamber and permitted to invade at 37°C for 48 h toward a lower reservoir containing medium and coated with Matrigel (BD Biosciences). The chambers were then fixed in 100% methanol for 30 min and stained with crystal violet for 10 min. The invasive cells which passed through the membrane were counted at ×200 magnification with five representative fields under a microscope. For migration, 2.5 × 10⁴ cells were added into the upper chamber without Matrigel and kept for 36 h under regular conditions. All the above assays were repeated in triplicate.

Scratch assay was performed to examine cell migration speed. Cells were incubated in 6‐well plate over‐night to yield monolayer confluence. By scratching with a pipette tip and photographing immediately (time 0) and 12 h' later, the distance migrated by the cell monolayer to close the scratch area during the time period was observed and measured. The ratio of the cell migration distance at 12 h to that at 0 h was analyzed as the migration index. The assay was carried out in triplicate and repeated three times.