Measuring Invasion Capacity of NPC Cells

For measuring the effects of UBE2T on invasion capacities, matrix-coated transwell invasion assay was performed as previously described [36 (link)]. Briefly, approximately 1×10⁵ NPC cells were suspended in serum-free medium and added to the top chamber of the 24-well, matrix-coated, transwell chamber system (Corning, Cambridge, MA, USA). Normal cell culture medium was added to the bottom chamber and cells were incubated for 24 hours at 37°C. The membrane was fixed and stained. Cells on the membrane were removed and the number of cells below the membrane were counted (5 random × 200 fields per well). For migration assay, the general transwell chamber was used instead of matrix-coated transwell chamber. Additionally, 2 μM AKT inhibitor (MK-2206 2HCl, S1078, Selleckchem, Houston, TX, USA) was used in the transwell analysis.