Figure 6. Expressing vimentin in MCF7 cells results in increased cell stiffness, cell motility, and directional cell migration.

A. Levels of vimentin overexpression in MCF7 cells were detected using Western blotting. MDA-MB 231 cells served as a positive control for vimentin protein expression. PSmOrange plasmid conjugated with vimentin was transfected into MCF7 cells. The molecular weight of vimentin was 57 KD, and the molecular weight of PSmOrange-vimentin shifted to 81 KD. Cell stiffness was analyzed using AFM indentation. Each bar represents mean ± SEM of 100 cells in each condition collected from three independent experiments. B. Representative images of wound healing assay. MCF7 cells were transfected with PSmVec as vector control and PSmVimentin. Cells were seeded for 18 hours until a monolayer was formed. The monolayer was then scraped using tips. Images were produced after scraping for 0 (space between red dashed lines) and 24 hours (space between yellow dashed lines). The quantitative data of wound healing is shown in the right panel. The wound healing percentage was normalized to PSmVec. C. Representative images of PSmVec and PSmVim time-lapse tracking for 9 hours. Cells were seeded at low density for 18 hours, and cell migration was recorded for an additional 9 hours. The upper panels showed the original cell position at 0 hours, and the middle panels showed the cell position after live cell tracking for 9 hours. Each line with a different color indicates the migration trace of each cell. All cell migration traces were placed in the same center in the tracking images. Cells measured for each condition: PSmVec: n = 24; PSmVim: n = 25. D. Quantitative data of directional migration. Left panels indicate distances, and right panels show the velocity of cell migration. Each bar represents mean ± SEM. ** indicates p < 0.01, and *** indicates p < 0.001 compared with the PSmVec control. E. and F. are the representative immunofluorescence images of vimentin overexpressing MCF7 cells. E. The left panels show the XY-Z projected images of overexpressing vimentin or vector control (red), microtubule (green), and merge images. The nucleus is blue. The right panel shows the microtubule distribution at the cell top and bottom for each condition. Scale bar: 20 μm. F. The left panels show the XY-Z projected images of overexpressing vimentin or vector control (red), actin (green), and merge images. The nucleus is blue. The right panel shows the actin distribution at the cell top and bottom for each condition. Scale bar: 20 μm.