CD133 and Colorectal Cancer Figure 1. Evaluation of CD133+ populations by flow cytometry. Eight CRC cell lines were tested and showed varying size of the population of CD133+ cells. (a) Sample flow cytometry images are shown for Caco2 (left panels, .95% positive cells) and SW837 (right panels). Analysis was performed using AC133/1 antibody (top panel) and gating was performed for each cell line using an isotype control (bottom panel), PMTLin 1 is photmultiplier tube linear 1 which indicates side scatter) (b) shows that in two cell lines there were no CD133+ cells identified whilst in two cell lines nearly all cells were CD133+. The remaining cell lines had varying size population of CD133+ cells. doi:10.1371/journal.pone.0010714.g001 either CD133 specific or scrambled control siRNA. Cell numbers were assessed on days 1, 3 and 5. For SW480 the sorted CD133+ and CD1332 populations were seeded at 104 cells per well and cell numbers assessed on days 1, 3, 5, 7 and 9. At least two independent experiments, each in triplicate, were performed. A methylene blue assay [21,22] was used to quantify numbers of adherent viable cells. Stauropsorine induced apoptosis Staurosporine was used to evaluate the resistance conferred by CD133 to exogenous apoptotic stresses. For HT29, each well of a 96 well plate (Costar) was seeded with either HT29CD1332 or HT29ssc cells 72 hours after transfection. Approximately 56104 cells were seeded per well and incubated with staurosporine (Sigma) at a final concentration of 8 mM for 24 hours. After this period, viable and apoptotic cells were measured. For SW480, each well of a 96 well plate was seeded with 105 cells of the sorted cells and staurosporine was added 24 hours later at a concentra- tion of 8 mM. After another 24 hours, the numbers of viable cells/ apoptotic bodies were assessed. The assay was performed in triplicate and repeated in at least two independent experiments. 2 dimensional (2D) and 3 (3D) dimensional colony forming assay The ability of isolated single CD133+ and CD1332 cells to form colonies was tested in both 2 dimensional (2D) culture and 3 dimensional (3D) culture. For 2D culture, 300 freshly sorted cells were seeded into individual wells of a 6 well plate and cultured for 14 days. The cells were then stained with methylene blue and colonies containing more than 20 cells were counted. The experiments were carried out in triplicate and on two occasions. 3D culture was performed to assess the colonogenic ability of the sorted populations in non adherent conditions. Cells (2500 from each population CD133+ and CD133-cells) were counted and resuspended as single cells in 0.7% DNA grade agarose (Sigma Aldrich).This was overlaid on a base of 1% DNA grade agar (Sigma Aldrich) and both top and base layers were mixed with 2X DMEM. Experiments were set up in triplicate and medium changed twice a week. After two weeks, the number of colonies that developed within each well was counted and visualized under a microscope after staining with 0.05% crystal violet for 1 hour and representative fields were photographed. For both 3D and 2D culture, percent colony forming efficiency (CFE) was calculated as follow, % CFE = (Number of obtained colonies/Number of cultured cells) X 100 [23]. Log transformed values were then used for statistical analysis. In vitro migration assay Transwell cell migration assays were performed using a Boyden chamber containing a polycarbonate filter with an 8 mm pore size (Costar). Culture medium (600 ml) supplemented with 20% FBS was added to the lower chamber and 2.56105 cells of the sorted populations were added into the upper chamber (in 100 ml of culture medium supplemented with 10% FBS). The number of cells migrating through the membrane was manually counted after 24 hours. Assays were performed in triplicate and on two separate occasions. Cell migration was also measured using a cell wounding assay performed in 6 well plates (Costar). Cells were grown to confluence and then starved for 24 hours in serum free medium. A sterile 200 ml pipette tip was used to create three separate parallel wounds and migration of the cells across the wound line was assessed after 24 hours. Photographs were taken using a charge- coupled device (CCD) camera (Canon, Japan) attached to the Figure 2. Splice variants of CD133. Cell lines and normal mucosal samples were tested for expression of the two main CD133 splice variants by RT- PCR with primers anchored on either side of the spliced out exon (exon 4). Sample data are shown which demonstrate only a single product was identified when PCR products were analysed on both (a) agarose gel and (b) the more sensitive Q-PCR technique using Sybr green as a reporter. M = size marker, nc = negative control, * represents DLD1. (c) Sequencing of the products showed that exon 4 was spliced out of the coding sequence thus only shorter splice variant (AC133-2) was being expressed. The sequence above the electropherogram is of AC133-1 with exon 4 shown between the lines. doi:10.1371/journal.pone.0010714.g002 PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10714