For the wound-healing assay, the A549 and H460 cells were seeded in an SPLScar Block cell culture dish (#201935, SPL, Pocheon, Republic of Korea). This was composed of a 500 um thick wall to artificially generate a cell-free gap. The cells were incubated for 24 h after the culture medium was immediately replaced with RPMI containing ebractenoid F. Using an Olympus light microscope, migrated cell pictures were observed and analyzed using ImageJ software (NIH).
For the trans-well assay, the A549 and H460 cells were seeded on upper chamber inserts (8 μm pore trans-well; Corning Inc., New York, NY, USA). The ebractenoid F-treated lung cancer cells were plated at 2.0 × 104 cells per well and incubated at 37 °C, 5% CO2, in a humidified incubator. The cells were then fixed with 4% formaldehyde for 5 min and permeated with 100% methanol for 15 min before being stained with 0.1% crystal violet for 20 min. In the upper chamber, non-migrated cells were removed with a cotton swab. Using an Olympus light microscope, migrated cell pictures were observed and analyzed using ImageJ software (NIH).
For the trans-well assay, the A549 and H460 cells were seeded on upper chamber inserts (8 μm pore trans-well; Corning Inc., New York, NY, USA). The ebractenoid F-treated lung cancer cells were plated at 2.0 × 104 cells per well and incubated at 37 °C, 5% CO2, in a humidified incubator. The cells were then fixed with 4% formaldehyde for 5 min and permeated with 100% methanol for 15 min before being stained with 0.1% crystal violet for 20 min. In the upper chamber, non-migrated cells were removed with a cotton swab. Using an Olympus light microscope, migrated cell pictures were observed and analyzed using ImageJ software (NIH).