Matrigel uncoated and coated transwell inserts

Matrigel uncoated and coated Transwell inserts (8 µm pore size; Millipore, Billerica, MA, USA) were used to examine the migration and invasion abilities of breast cancer cell in vitro. Briefly, a total of 5×10⁴ transfected cells were suspended in 150 µl serum-free DMEM and added to the upper chamber of the Transwell chambers, and 600 µl of 20% FBS containing DMEM was loaded in the lower chamber. After 48 h, the non-migratory cells on the upper side were removed using a cotton swab, cells located on the lower surface of the chamber were fixed in 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet for 15 min. The mean number of migrated or invaded cells was counted by averaging the numbers of cells in 10 fields in both inserts under a microscope (Olympus, Tokyo, Japan). All experiments were performed in duplicate and repeated at least three times.

The lncRNA-MEG3-transfected HGC-27 cells were cultured in Matrigel-coated and -uncoated Transwell inserts (8 µm pore size; Merck KGaA) for the invasion and migration assays, respectively. HGC-27 cells (1×10⁴ cells/well) with 150 µl serum free DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) was placed into the upper chamber and DMEM with 5% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.) was placed in the lower chamber. Following 24 h incubation at 37°C, HGC-27 cells in the upper chamber were removed with a cotton swab and those in the lower chamber were fixed in 4% paraformaldehyde for 15 min at 37°C, stained with 0.1% crystal violet dye (Sigma-Aldrich; Merck KGaA) for 20 min at 37°C and counted at three randomly selected views using an Olympus BX51 light microscope (Olympus Corporation, Tokyo, Japan).

Patient-derived ovarian cancer cells were treated with NK cells (effector:target ratios=15:1) for 24 h at 37°C and non-treated cells were used as control. For the invasion assay, NK-treated cells were suspended at a density of 1×10⁵/ml cells in 500 µl serum-free DMEM. Matrigel-coated and uncoated Transwell inserts (8 µm pore size; Merck KGaA, Darmstadt, Germany) were used to evaluate cell invasion and migration, respectively. The ovarian tumor cells (2×10⁵) were then subjected to the tops of BD BioCoat Matrigel Invasion Chambers (BD Biosciences, Franklin Lakes, NJ, USA) for 48 h at 37°C according to the manufacturer's protocol. For the migration assay, patient-derived ovarian cancer cells were treated with NK cells and PBS for 48 h using a control insert (BD Biosciences) instead of a Matrigel Invasion Chamber. DMEM and DMEM with 5% FBS was plated in the upper and lower chambers, respectively. Cells were then fixed in 4% paraformaldehyde for 15 min at 37°C and stained with 0.1% crystal violet dye (Sigma-Aldrich; Merck KGaA) for 20 min at 37°C. Tumor cell invasion and migration was counted in at least three randomly stained fields under a light microscope (Olympus Corporation, Tokyo, Japan) for every membrane (magnification, ×40).

For the migration and invasion assays the lnPTENP1- or vector-transfected Mg63 cells were placed into the upper chamber of Transwell plates with non-coated membranes at a density of 1×10⁴ cells/well with 150 µl serum-free Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.). Matrigel-coated and uncoated Transwell inserts (8 µm pore size; Merck KGaA) were used to evaluate cell invasion and migration, respectively. The cells were incubated in DMEM with 5% FBS (both Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h at 37°C and then the Mg63 cells were fixed in 4% paraformaldehyde for 15 min at 37°C and stained with 0.1% crystal violet dye (Sigma-Aldrich; Merck KGaA) for 20 min at 37°C. The cells were removed with a cotton swab and counted at three randomly selected views using a light microscope (BX51; Olympus Corporation, Tokyo, Japan) at a magnification of ×40.