Transwell plates with 8 μm pore membranes

Transwell plates with 8-μm pore membranes (353097, Corning Life Sciences, Corning, NY, USA) were used to analyze cell migration and invasion. For the migration assay, Pancreatic tumor cells (KPC and BxPC-3: 5 × 10⁵/well) in serum‐free medium were added to the upper chamber. In total, 500 μL of complete medium containing 10% FBS was added into the lower chamber. DMSO and the USP8 inhibitor (1 μM) were added to the upper chamber after cells attached. After incubation for 24 h, the migratory cells were formalin-fixed and crystal violet (0.1%) stained. Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Bethesda, MA, USA) was used to count the migratory cells. For assays of cell invasion, the Transwell chambers were pre‐coated with Matrigel (1 mg/ml Matrigel matrix, BD Biosciences, Franklin Lakes, NJ, USA). The remaining steps were similar to those described in the migration assay.

The CSE-transformed HBE cells were suspended in serum-free medium at a density of 1 × 10⁵ cells/mL. Subsequently, 200 μL of cell suspensions were added to the upper chambers of Transwell plates with 8-μm pore membranes (Corning, Inc., Corning, NY, USA) with 35 μL of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). MEM media (500 μL) supplemented with 10% FBS was added to the lower chambers. After incubation for 24 h at 37°C, the cells on the upper surfaces of the microporous membranes were removed with cotton swabs, whereas cells on the lower surface of the membranes were fixed with 4% paraformaldehyde, stained with crystal violet solution for 30 min, and washed twice with PBS. Images of the stained cells from five selected views were captured under a microscope (high-power field), and the numbers of cells that migrated through the membranes were averaged. To assess the capacity for migration of transformed HBE cells, transfected cells (5 × 10⁴/100 μL) were added to upper chambers without Matrigel. MEM medium containing 10% FBS was added to the lower chambers. Cells were incubated for 24 h at 37°C. Migrating cells were fixed, stained, and calculated.