TIMELESS inhibits breast cancer cell invasion and metastasis by down-regulating the expression of MMP9

摘要

The effect of TIMELESS on the cellular motility of breast cancer cells. a Phase-contrast microscopic images of the human breast cancer cell lines. MCF-7, T47D and ZR-75*30. b Western blot analysis of TIMELESS expression in the breast cancer cells. c, d Scratch wound-healing assay assessing the effects of TIMELESS on the motility of breast cancer cells. e, f Transwell migration and invasion assays evaluating the effects of TIMELESS overexpression (upper) or knockdown (lower) on the cellular motility and invasion ability of breast cancer cells. Cell migration and invasion assays were performed in 24-well chambers without or with Matrigel. Cells were transfected with FLAG-TIMELESS and control vector (Ctrl) or shTIMELESS#1 and control vector (shCtrl) and then plated in the upper chamber. After 24 h of incubation, the migrating and invading cells on the lower surface of the filter were stained and counted. g, h Immunofluorescence assays accessing the effects of TIMELESS overexpression (left) or knockdown (right) on the F-actin organization of breast cancer cells. 2 × 105 cells were cultured on the cover glass in each new 35 mm cell culture plate. F-actin immunofluorescence was examined and photographed using a Nikon TE2000-U microscope after 24 h-transfection with corresponding plasmids. F-actin was visualized with tetraethyl rhodamine isothiocyanate (TRITC). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Each bar represents the mean ± s.d. from three independent experiments. “*”, p < 0.05; “**”, p < 0.01