Figure 4.

IL-6 and TNF-α cotreatments increased colonosphere formation and cell invasion whereas withaferin A decreased the sphere formation and cell invasiveness. (a) Colonosphere formation assay. DLD1 cells were cotreated with IL-6 and TNF-α for 24 hours, then subjected to the three-dimensional culture condition. Withaferin A (10 μM) was pretreated with IL-6 and TNF-α, then applied to three-dimensional culture. Representative images were taken after 7 days. Scale bar represents 50 μm in length. (b) Quantitative representation of colonospheres formed in IL-6 and TNF-α cotreatments in the absence and presence of withaferin A. ^∗p < 0.05, ^∗∗p < 0.01 versus untreated control. (c) Boyden chamber assay. Cell invasiveness was examined by employing Boyden chamber assay. Human colorectal cancer DLD1 cells were subjected to Boyden chamber cell invasion assay. Cells were treated with IL-6 and TNF-α in the absence or presence of withaferin A. Assays were done for 48 hours. Representative images were taken. Scale bar represents 50 μm in length. (d) The cell invasion assay was quantitatively measured in graphic representation. ^∗p < 0.05, ^∗∗p < 0.01 versus untreated control.