CD133 and Colorectal Cancer Figure 6. Effect of CD133 on cell motility. Transwell migration was measured after knockdown of CD133 in HT29 and sorting of SW480 into CD133+/2 populations. Significantly fewer HT29CD1332 cells migrated across the membrane than HT29ssc cells (Figure 6a, p = 0.04). Conversely, larger numbers of sorted CD133+ cells migrated than CD1332 cells (Figure 6b, p = 0.03). A wounding assay was also undertaken and gene knockdown was associated with marked delay in closure of the wound which was visiually perceptible after only 24 hours (Figure 6c) and statistically significant (p,0.001). doi:10.1371/journal.pone.0010714.g006 were obtained indicating that high levels of CD133 conferred staurosporine resistance. Fewer viable HT29CD1332 cells were present than HT29ssc cells (Figure 5a, p = 0.01); conversely greater number of viable cells were present in the sorted CD133+ population than the CD1332 population(Fig. 5b, p = 0.008). There was however no difference between cells when exposed to DMSO alone. Another features of ‘‘stemness’’ is the ability to form colonies. This was tested by growing single cells in soft agar for two weeks (3D culture) and 2D culture and the sorted CD133 + and CD133 2 cells were compared. In both cases, colonies were visible after two weeks but the number of colonies was significantly greater in CD133 + cells (P,0.0001, and P,0.003) in 2D and 3D culture respectively (Figure 5c and 5d). Association of CD133 expression with cell migration Comparative analysis of cell motility between cells with high and low CD133 expression was tested by transwell migration assays. Both experimental conditions produced concordant results. Significantly fewer HT29CD1332 cells migrated across the membrane than HT29ssc cells (Figure 6a, p = 0.04). Conversely, significantly larger numbers of CD133+ cells migrated than CD1332 cells (Figure 6b, p = 0.03). A cell wounding assay was also used following gene knockdown which showed that the wound edges were significantly closer in HT29ssc cells than in HT29CD1332 cells (p,0.001) thereby confirming the relationship between high CD133 expression and increased cell motility (Figure 6c). PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10714