Matrigel coated chambers with 8 μm pores

The invasion of cells was assessed using Matrigel-coated chambers with 8 μm pores (BD Biosciences, Franklin Lakes, NY, USA). Briefly, hepatoma cells (1 × 10⁵) were seeded in serum-free medium and were allowed to translocate toward complete media supplemented with 10 % fetal bovine serum after depleted of UPF1. The cells that had invaded through the membrane to the lower surface were fixed, stained and counted after 24 h. HCC cells (1 × 10⁶ cells/well) were treated with the indicated reagents, and wounds were made using a 100 μl plastic pipette tip. The size of the wound was measured after 24 h of wound formation and photographed.

Approximately 1 × 10⁵ PDAC cells treated with different reagents were suspended in serum-free medium and seeded in either Matrigel-coated chambers with 8-μm pores (BD Biosciences, Franklin Lakes, NY, USA) for migration assays or in chambers (Corning Costar, NY, USA) that were not coated with Matrigel for invasion assays. For both assays, medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 12 h, the cells that had invaded through the membrane to the lower surface were fixed with methanol, stained with 0.5% crystal violet solution and counted in 5 random fields of view (200×). RhoA pathway specific inhibitor CCG-1423 [32 (link)] was purchased from selleck and used as recommended.