HCT-116 and LoVo cells were xed for 30 minutes with 4% paraformaldehyde at room temperature, permeabilized with Triton X-100 (0.5%) for 15 min, then blocked with 5% BSA solution for 1 h. The cells were incubated with primary antibodies for 1 h, following by incubating with secondary antibodies at room temperature for 1 h. After incubation, nuclear was labeled with DAPI for 5 minutes. Finally, cells were tested with a DMI3000B inverted microscope (Leica, Germany). Transwell assay for migration HCT-116 and LoVo cells were inoculated into the upper chamber of transwell plate and cultured in 600 μl 1640 or F12K medium with 10 μg/ml bronectin, medium containing 15% FBS was added in the lower chamber of transwell plate. Then different concentrations of Tan IIA were added into the upper chamber and incubated for 48 h. Migrated cells were detected by crystal violet staining, and observed by using DMI3000B inverted microscope (Leica, Germany). Five random views were selected to count the migrated cells.
Wound-healing assay HCT-116 or LoVo cells were seed into 6-well plates for 24 h, then were created an arti cial scratch wound using a 20μl pipette tip and detached cells were removed by washing with PBS three times. After 48 h incubation, cell migration was photographed using inverted microscope and evaluated by measuring the difference in wound width.
Wound-healing assay HCT-116 or LoVo cells were seed into 6-well plates for 24 h, then were created an arti cial scratch wound using a 20μl pipette tip and detached cells were removed by washing with PBS three times. After 48 h incubation, cell migration was photographed using inverted microscope and evaluated by measuring the difference in wound width.