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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_24061_sm_SuppFig1.tif10KSupporting Information Figure 1. FACs analysis of CD44 expression in colon tumor cell lines.
IJC_24061_sm_SuppFig2.tif90KSupporting Information Figure 2. Trypan blue cell count analysis of LSI74T cells sorted into CD44I1 and CD44-; populations and cultured for 2 days under standard cell culture conditions. Values are the mean ± standard error of 5 wells counted for each population. Difference was statistically significant (p=0.005) by student's t-;test. b) Measurement of ATP in freshly sorted CD44hi, CD44-; and live primary tumor xenograft cells at day 0 (immediately after sort, grey bars), and after 2 days incubation in DMEM-10%FBS (black bars). Experiment was repeated twice with similar results.
IJC_24061_sm_SuppFig3.tif224KSupporting Information Figure 3. Evaluation of reactive oxygen species (ROS) in CD44 colon tumor cells. A, B) CD44hi and CD44- colon tumor cells have similar levels of ROS. Freshly sorted CT3x primary colon tumor xenograft cells were loaded for 40 minutes with DCF, then washed and incubated with anti-CD44 APC antibody. Dot plots show CD44 staining in the absence of DCF (A) or in combination with DCF (B). Costaining tumor cells with CD44 and DCF indicates similar levels of ROS production in both the CD44hi (upper right quadrant) and CD44 (lower right quadrant) cells. C) Pre-treatment with the anti-oxidant N-acetylcysteine (NAC) significantly reduced levels of ROS (control no-DCF, grey fill; DCF alone, black line; NAC + DCF, grey line). Experiments were performed twice with similar results in primary colon xenograft and tumor cell lines. D) No significant differences in cell growth were observed in CD44hi and CD44 sorted CT3x tumor cells treated for 48 hours with 0, 1, 5 and 10mM NAC. E) CD44 sorted LS174T colon tumor cells were seeded in triplicate in soft agar ± 5mM NAC. CD44hi cells (black bars) formed more colonies than CD44- cells (grey bars) as previously demonstrated. NAC treatment did not significantly alter the growth advantage of CD44t cells in soft agar, and instead increased colony growth —‘2-fold in both CD44hi and CD44- cells. Similar results were also observed in SV’d820 colon tumor cells (data not shown).
IJC_24061_sm_SuppFig4.tif445KSupporting Information Figure 4. A) FACs plot showing BrdU labeled primary tumor xenograft cells from CT3 (left) and CT5 (right) stained with anti-CD44 PE followed by intracellular staining with anti-BrdU APC. CD44- BrdU+ cells are in the R4 gate, while CD44hi BrdU+ cells are in the R5 gate. B) Cell cycle analysis comparing CD44hi and CD44 sorted CT5 primary colon tumor xenograft cells. Similar results were seen for CD44 sorted cells from CT2 and CT4 tumor xenografts.
IJC_24061_sm_SuppFig5.tif619KSupporting Information Figure 5. FACs analysis of the CT5 primary colon tumor xenograft passaged through two serial transfers of 1000 CD441 cells. Top row shows CD44 stained samples, and bottom row shows matched isotype controls for each sample used to gate the CD44 population. Bottom left plot shows staining two-color analysis of CT5 stained with anti-ESA FITC and mouse lineage-PE, indicating that all non-mouse human tumor cells are ESA+.
IJC_24061_sm_SuppFig6.tif4052KSupporting Information Figure 6. Sections from the CTS colon tumor sample either freshly isolated (primary tumor, left column), after mouse xenograft passage 2 (middle columns) and from a tumor derived from 100 CT3 CD44hi cells (right columns) illustrate that tumor histology by H&E staining (top panels, 200x), and common tumor markers such as CEA (middle panels) and the human epithelial specific antigen recognized by the AE1/3 antibody cocktail (bottom) are maintained from the primary tumor to the xenograft passaged tumors.
IJC_24061_sm_SuppFig7.tif4KSupporting Information Figure 7. Analysis of ATP levels in CD44 and CD44/ALDH double positive primary colon tumor xenograft cells.
IJC_24061_sm_SuppFig8.tif223KSupporting Information Figure 8. A. FAGs analysis of CD44 and IGF-1R expression in primary colon tumor xenograft cells. Bar chart on right compares the mean fluorescence intensity of IGF-IR staining on CD44hi versus CD44- cells from two different xenograft tumors. B. FAGs analysis of CD166 and CD44 expression in CT4 primary xenograft tumor cells.
IJC_24061_sm_SuppTable1.tif65KSupporting Information Table 1. CDI 33 and CD133/CD441 expression on colon tumor samples.
IJC_24061_sm_SuppTable2.tif387KSupporting Information Table 2. In vivo tumorigenicity of colon tumor cells sorted by CD133 and ABCG2.

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