Transwell Assay for Invasion Analysis

The influence of LINC01013 on invasion ability in vitro was determined by Transwell assay (Falcon BD, Franklin Lakes, NJ) using LINC01013-depleted KARPAS-299 or LINC01013-overexpressed SR-786 cells, as described previously^{15 (link)}. Briefly, cell density was adjusted to 10⁵ cells/ml, and 100 μl of the suspension was seeded into upper chambers of the Transwell plate, either coated (invasion) or not coated (migration) with Matrigel (Becton-Dickinson). For both assays, the pore size of the upper chamber was 8 mm. The medium in the upper chamber was serum-free DMEM, and the lower chamber contained DMEM supplemented with 20% FBS, included as a chemoattractant. After incubation for 24 h at 37 °C, cells traversing the filter from the upper to lower chamber were stained with crystal violet and counted. Experiments were repeated at least three times.