8.0 μm pore size polycarbonate membranes

For the wound healing assays, PC cells were plated in 6-well plates and cultured at 37 °C for 24 h. Wounds were made in the monolayer of the cells using a standard 200-μl pipette tip. Cells were washed to remove debris and incubated in DMEM without FBS at 37 °C. Images were taken at 24 h after wounding. The wound area was measured, and the percentage of wound healing was estimated by using ImageJ software (NIH, Bethesda, MD, USA). Cell invasion assays were conducted using 24-well transwell chambers with 8.0-μm pore size polycarbonate membranes (Corning Inc., Corning, NY, USA). Then, cells (5 × 10⁴) were seeded on the top side of the membrane pre-coated with Matrigel (Corning Inc.). After incubation, the cancer cells inside the upper chamber were removed with cotton swabs. The invaded cells on the lower membrane surface were then fixed and stained with a 5% crystal violet solution. Three images of ten random fields of each membrane were captured, and the number of migratory cells was counted. The means of triplicate assays for each experimental condition were used. The migration and invasion index is defined as the ratio of the experimental group to the control group.

Cell migration of DLD-1 was further evaluated using the transwell migration assay. Cells were transiently transfected with Uc160 or Uc346 or vector for 20h and left in full growth medium post-transfectionally for 24h. Cells were suspended in serum-free culture medium and 1×10⁵ cells were loaded onto the top of Transwell chambers equipped with 8.0 μm pore-size polycarbonate membranes (Corning Inc., NY, USA) and allowed to migrate towards 10% FBS medium for 24h. Migrated cells were fixed with Carson's buffer and stained with Giemsa. Images were captured at a magnification of 40x using an Axiocam ERc 5s camera (Carl Zeiss Microscopy LLC) mounted on an inverted microscope (Axiovert 40 CFL, Carl Zeiss Microscopy LLC) and the average number of migrated cells was calculated using five different fields.