Tscratch software version 1

The anti-migration effects of ACA and its analogs were determined using the wound-healing assay. MDA-MB-231 cells were seeded in six-well plates and cultured until 80% confluent. After an overnight cultivation, complete medium was then replaced by serum-free medium treated with 1 μg/mL mitomycin-C, and the cells were further incubated at 37°C for 2 h to stop cell proliferation. A vertical scratch was made using a sterile pipette tip across the well, and cell debris was removed by washing with 1× phosphate-buffered saline. Cells were treated with control solvent or ACA and its analogs at their 20% inhibitory concentrations (IC₂₀; Table S1) in serum-free medium. IC₂₀ were used instead of IC₅₀, because IC₂₀ were less toxic on the cells. Images of wounded cells were captured using an inverted fluorescence microscope, Nikon Eclipse TS-100 (Nikon Corporation, Tokyo, Japan), at 0 and 24 h after wounding. The distance between two sides of the wound was analyzed using Tscratch software, version 1.0 (The Mathworks, Natick, MA, USA).

The cancer cells treated with taraxasterol were performed by in vitro scratch test due to analysis of migration ability. Prostate cells were cultured in a four-well plate with 70,000 cells per well and allowed to form a monolayer. Then, the monolayers were scraped using a sterile pipette tip and rinsed with PBS. The IC₅₀ concentration (114.68 ± 3.28 μM) of taraxasterol was added to the plates and incubated for 24 h for the next steps. Imaging was then performed using a reverse microscope. Image analysis was performed using TScratch software version 1.0 (Math Works Inc., MA and USA). Each group (prostate cancer and taraxasterol treatment) was evaluated in three replications.