Figure 4.

Effect of caffeine on anchorage-independent growth and CSC-like phenotypes of NCI-H23 cells. (a) The growth of cells in anchorage-independent conditions was investigated with soft agar colony-formation assay. The cell was cultured in soft agar containing various concentrations of caffeine (0–500 µM) for 7 days, then cell colonies were captured under an inverted microscope (b) Histogram shows the percentage of colony number. (c) The dot plot represents the percentage of colony size. (d,e) The CSC-like phenotypes were examined using a single 3D spheroid-formation assay. The secondary CSC-enriched spheroids were treated with 0–500 µM of caffeine, and the enlargement of spheroids was investigated at 0, 3, and 7 days. (f) The secondary spheroids were plated onto matrigel and treated with caffeine at concentrations of 0–500 µM. Cell migration away from the spheroid was imaged with an inverted microscope at 0, 3, 5, and 7 days. (g) The relative spheroid migration value was calculated from areas between the blue and red lines. Data are expressed as mean ± SD (n = 3). * p < 0.05 compared with the non-treated control.