Cell Migration Assay

To determine the capability of cell migration, we used both wound healing and Boyden chamber Transwell assays. For the wound healing assay, a silicon gap insert (Ibidi GmbH, Martinsried, Germany) was placed in a 12-well culture plate to create a cell-free space of 400 µm in width between 2-well chambers. MDA-MB-231 cells (2 × 10⁶ cells/well) were seeded into each chamber of the culture insert. After 24 hours of incubation, the silicon insert was removed and the cells were washed with phosphate-buffered saline to exclude nonadherent cells and debris. Then, cells were treated with 10 µg/mL ANR, ANS, or ANL for indicated times. For the Boyden chamber assay, a Transwell insert with 8.0 µm pore of polyethylene terephthalate polyester membrane (Corning Inc, Corning, NY) can allow cells to migrate through the pore. The MDA-MB-231 cell suspension (5 × 10⁴ cells/well) culture in FBS-free RPMI-1640 medium containing DMSO vehicle or 10 µg/mL ANR, ANS, or ANL were seeded into each top insert. Cells were allowed to migrate to the bottom of the membrane, which was immersed in 10% FBS medium for 16 hours and then fixed with 70% ethanol. Nonmigrated cells were scraped off with a cotton bud, and the migrated cells were stained with 0.1% crystal violet. A digital CCD camera mounted on an inverted microscope obtained all images (Axiovert 40, Zeiss, Thornwood, NY), and the wound distances and cell numbers were calculated, respectively, for the wound healing assay and the Boyden chamber assay using AxioVision Release 4.8 software (Carl Zeiss AG, Oberkochen, Germany).