Figure 5. Effect of RUNX1 knockdown on SKOV3 cell migration and invasion. (A) Migration was assessed using Boyden chamber assay. Cells from the shRNA-RUNX clone (sh-cl4) and the control clone (ctrl3) were seeded into the upper chambers in 0.1% FBS containing medium at a density of 2.5 × 10⁴ per well, and 600 μl of 1% FBS-containing medium was placed in the lower chamber as a chemoattractant. After 24 h at 37°C in 5% CO₂, the cells were fixed with cold methanol and stained with blue trypan solution. Migrated cells on the underside of the filter were photographed and counted by phase contrast microscopy. (B) Cell invasion was assayed in a similar way, as the upper chambers were coated with Matrigel. Here, NIH3T3 conditioned medium was added in the lower chamber as a chemoattractant (see “Materials and Methods” for details). All experiments were performed in triplicate. For each experiment, cell number was calculated as the total count from 10 random fields per filter (at magnification ×40). Differences between shRNA-RUNX1-transfected and vehicle-transfected SKOV3 cells were determined by a Student’s t-test, where p < 0.05 was considered significant.