Figure 3. Knockdown of CD133. HT29 cells were transfected with either CD133 specific siRNA (HT29CD1332) or scrambled control ((HT29ssc). Western blotting confirmed the loss of CD133 expression by gene knockdown. b-Actin shows equal protein loading. doi:10.1371/journal.pone.0010714.g003 inverted phase-contrast microscope at a power of X40. The distance between the edges was measured at 6 equally distributed points using ImageJ software [33] and then analysed using a two tailed t-test. Experiments were repeated on two separate occasions. Statistical analysis Statistical analysis was undertaken using SPSS 13.0 Software. All evaluations were done using unpaired two tailed Student’s T- test. For studies involving cell and colony counting, cell numbers were analysed. For studies with a numerical fluorescence output, the raw fluorescence values were used. Results CD133 expressing populations in cell lines The size of the CD133+ population was tested in 8 CRC cell lines by flow cytometry and in each case approximately 500 000 cells were analysed. In two cell lines (DLD1 and SW837), CD133+ cells were not detectable. The size of CD133+ population was found to be .95% in two cell lines (Caco2 and HT29). In the remaining cell lines, a bimodal population was present with a CD133+ population ranging from 32–64% (Figure 1). Splice variants of CD133 in CRC cell lines and normal mucosa Expression of the two main splice variants of CD133 (AC133-1 and AC133-2) was tested by RT-PCR in both the cell lines and samples of normal mucosa. Only one product was seen on agarose gels which, on sequencing, was found to be the shorter AC133-2 splice variant from which exon 4 is spliced out (Figure 2a and 2c). However, shorter PCR products undergo preferential amplifica- tion and it is possible that larger products may be missed by both agarose gel electrophoresis and sequencing (especially if present at low levels). The analysis was refined by performing PCR using SYBR green as a reporter dye. Evaluation of the dissociation curve showed the presence of a single PCR product only (Figure 2b). Knockdown of CD133 HT29 has a high level of CD133 expression. Cells were transfected with either CD133 specific or scrambled control siRNA and were tested 72 hours post transfection. Western blot CD133 and Colorectal Cancer Figure 4. Effect of CD133 on proliferation. Cell proliferation was evaluated after knockdown of CD133 in HT29 (Figure 4a) and sorting of SW480 into pure CD133+ and CD1332 populations (Figure 4b). A time course assay was performed over several days with no association seen between CD133 and cell numbers. doi:10.1371/journal.pone.0010714.g004 showed that the protein was virtually undetectable in the when CD133 specific siRNA was used. In contrast, transfection of the control siRNA has no effect on protein expression (Figure 3). Association of CD133 expression with cell proliferation We compared the proliferation rate between cells with high and low CD133 levels using two experimental approaches. In HT29 the CD133 protein was knocked down and cell numbers monitored for 5 days (Figure 4a) whilst SW480 was sorted into CD133+/CD1332 populations and cell numbers were monitored over 9 days (Figure 4b). Both experiments showed the same results i.e. there was no significant difference between cells with high or low CD133 expression. Similarly, counts of floating apoptotic bodies over this period did not show any difference (data not shown). Association of CD133 expression with staurosporine induced apoptosis and colony formation A feature that may be expected in stem cells is resistance to apoptosis following exogenous stress. Both experimental approaches were used to test the effect of CD133 levels on resistance to apoptosis when exposed to staurosporine for 24 hours. Concordant results PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10714