The effect of Timosaponin A-III on proliferation was determined by an MTT assay. Migration and Invasion assays were carried out as described [29 (link),30 (link)]. Briefly, MDA-MB-231 and MCF7 cells were plated in 24-well Corning Transwell Migration and BioCoat Matrigel® Invasion chambers, treated with Timosaponin A-III for 72 hours. 50,000 cells in 0.2 ml medium were added to the top chambers, and 1 ml DMEM was added to the bottom wells. After incubating overnight at 37°C, cells were fixed with methanol at −20°C, and stained with Diff-Quik stain (Dade Behring, Deerfield, IL). Unmigrated cells were removed from the top of the well, migrated cells were counted, and images were taken in multiple fields with a Nikon Eclipse Ti microscope under 10× magnification. For wound healing assay, MDA-MB-231 cells were cultured to confluence in 6 well plates and wound was made using a pipette tip and images were taken. Cells were either mock treated or treated with Timosaponin A-III (2 and 4 µM) and imaged again after 12 hr and 24 hr using a Nikon Eclipse Ti microscope under 10× magnification.