Fig. 2
Salinomycin reduces migration and invasion of murine colorectal cancer cells. To visualize tumor cell migration 0.5 × 106 MC38 (a) and CT26 (b) cells were cultured in 6-well plates until confluence. A scratch was created in the middle of the monolayer and cells were treated with 1 μM 5-FU or increasing concentrations of Sal (1 μM, 2 μM, 5 μM and 10 μM). Cell migration was assessed by phase-contrast microscopy and images were captured at the beginning of treatment and after 24 and 48 h at a magnification of 100. The open wound area after 48 h in cultured MC38 (c) and CT26 cells (d) was calculated and displayed as a summary of 3 independent experiments as mean ± SD; * p < 0.001 compared with control. For transwell-analysis of tumor cell migration 1 × 105 MC38 or CT26 cells were seeded in 6-well plates equipped with a transwell insert and exposed to 1 μM 5-FU, increasing concentrations of Salinomycin (1 μM, 2 μM, 5 μM and 10 μM) and a combination of 5-FU and Salinomycin. After 48 h membranes were stained with crystal violet solution and migrated cells were isolated from the lower side of the membrane and quantified by ELISA reader (e + f). To assess sustained effects of Salinomycin on tumor cell migration, the cells were treated for 48 h as indicated above and further cultured for another 48 h after lapse of the agent (g). Alternatively, MC38 and CT26 were cultured in Matrigel-coated transwell inserts. After 48 h or further incubation for another 48 h with fresh culture medium the number of invasive migrated cells was quantified as described above (h-j). Results are shown as representative images of stained membranes at a magnification of 100 or as summary of at least 3 independent experiments as mean ± SD; * p < 0.05 and ** p < 0.001 compared with control