Bz analysis application

143B cells were plated at a density of 5.0 × 10⁵ cells/well in 6-well plates and after 5 h, the media were changed to DMEM containing SK-216 (0, 25, or 50 μM). The next day, confluent cell monolayers were scratched using a sterile 1000 μL pipette tip using a CELL Scratcher^TM scratch guide (IWAKI, Tokyo, Japan). The wounds were captured immediately upon being scratched. After about 30 h, the wounds area was measured using software (BZ-analysis Application, Keyence, Osaka, Japan).

For the detection of E-cadherin or α-SMA, cells were fixed in ice-cold methanol for 10 min and treated with a rabbit polyclonal antibody against E-cadherin (1:150; sc-7870; Santa Cruz Biotechnology, Santa Cruz, CA) or α-SMA (1:100; ab5694; Abcam, Cambridge, UK) for 1 hour. The bound primary antibodies were detected using Zenon Alexa Fluor 488 (1:500; Life Technologies). For the detection of vimentin, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with phosphate-buffered saline (PBS) containing 0.1% TritonX-100 for 5 min. Thereafter, the cells were exposed to mouse Anti-vimentin eFluor 615 (1:100; eBioscience, San Diego, CA) for 1 hour. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Life Technologies). Digital images were captured on the at least six fields per sample with the same exposure time for each marker. The degrees of expression for E-cadherin, α-SMA, and vimentin were quantified by measuring fluorescence intensities of Alexa Fluor 488 or eFluor 615 per each field using software (BZ-analysis application, Keyence).