Eclpse 80i system

To observe the effects of lncRNA UBE2CP3 on cell migration and invasion, trans-well experiments were performed by using an 8-μm pore trans-well chamber (BD Biosciences, MA, USA) with/without Matrigel (BD Biosciences). In the invasion assay, a trans-well chamber was placed into a 24-well plate and coated with Matrigel. In both trans-well assays, cells were suspended in serum-free medium and were allowed to migrate toward the complete media supplemented with 10% fetal bovine serum after over-expression or knockdown of lncRNA UBE2CP3. After 24 h incubation at 37°C, migrated cells were fixed with 100% methanol for 30 min. Non-migrated cells were removed by cotton swabs. Next, the cells on the bottom surface of the membrane were stained with 0.1% crystal violet for 20 min. Cell numbers in 5 random fields in each replicate were counted using microscope (NiKon ECLPSE 80i system; magnification: × 200). Each assay was performed at least three times.

For the transwell migration assay, 10⁵ cells in 100 ml of serum-free DMEM media were triplicate seeded in each fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning). 600 μl of 10% NCS in DMEM was added to the bottom chamber. SW480 and SW620 cells were incubated at 37°C for 12 h. The inserts were washed twice with prewarmed PBS. Cells adhered on the lower surface were fixed with 100% methanol at RT for 15min and stained with hematoxylin for 15min. Cell numbers in six predetermined fields in each replicate were counted under the microscope (Nikon ECLPSE 80i system; ×200). All assays were independently repeated at least for three times. Cell invasion assays were performed as the migration assay except the transwell membrane was precoated with 24mgml⁻¹ Matrigel (R&D Systems) and the cells were incubated for 24 and 18 h, respectively.