Additional file 4: Figure S3. of Salinomycin inhibits metastatic colorectal cancer growth and interferes with Wnt/β-catenin signaling in CD133+ human colorectal cancer cells

Combined treatment with Salinomycin and 5-FU reduces migration and invasion of murine CRC cells and induces necrotic cell death. 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 Salinomycin (1 μM, 2 μM, 5 μM and 10 μM) and 1 μM 5-FU. 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 Salinomycin (1 μM, 2 μM, 5 μM and 10 μM) and 1 μM 5-FU. 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). 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 (G + H). 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. For detection of cell death 0.5 × 106 MC38 (I) or CT26 (J) cells were seeded in 6-well plates and grown until confluence following exposure to Salinomycin (1, 2, 5 and 10 μM) and 1 μM 5-FU for 24 h. Detection of cell death was performed using AnnexinV-FITC and PI staining and cells analyzed by flowcytometry. Results are displayed as representative dot blots or as summary of at least 3 independent experiments; ** p < 0.001 compared with control. (ZIP 370 kb)