Figure 3.

The effect of machilin D on cell growth and mammosphere formation. (A) MDA-MB-231 cells were treated with machilin D for 24 h in a culture medium. The cell growth assay using machilin D was measured with an EZ-Cytox kit. (B) Breast cancer MCF-7 cells were treated with various concentrations of machilin D for 24 h in a culture medium. The cell proliperation of the MCF-7 cells was measured with an EZ-Cytox kit. (C,D) Machilin D inhibits the formation of mammospheres. For the establishment of mammospheres, 1 × 10⁴ MDA-MB-231 cells and 4 × 10⁴ MCF-7 cells were seeded in ultralow 6-well plates using a CSC culture media. The mammospheres were incubated with increasing concentrations of machilin D or DMSO for seven days. Images showing representative mammospheres were obtained by microscopy (scale bar: 100 μm). The mammosphere formation efficiency (MFE) was examined. (E) Effect of machilin D on the migration of the breast cancer cells. The migration with/without machilin D was photographed at 0 and 24 h (scale bar: 100 μm). (F) Transwell assays were performed to determine the cell migration (without Matrigel) and invasion (with Matrigel) of the MDA-MB-231 cells exposed to machilin D (scale bar: 100 μm). (G) Machilin D inhibits the colony formation of the cancer cells. The cancer cells were incubated in 6-well plates and treated with machilin D. Representative data were collected. The data from triplicate experiments are represented as the mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001.