Lentiviral transduction of CoCSC To assess clonality using lentiviral insertion site analysis, or introduce shRNA targeting ALDH1A1 or Luciferase, UM-C4 and UM-C6 tumor cells were transduced in vitro over a 3–4 day period. Lentiviral vectors used for these studies contained either an eGFP- Luciferase fusion gene (pLOM65) for clonality studies, or ALDH1A1 (pLOM205) or a control Luciferase (pLOM145) targeted shRNA embedded in the eGFP mRNA; each of which is driven by the CMV promoter. Cells were isolated by FACS based on GFP fluorescence and transplanted into mice. For clonality studies, pLOM65-transduced cells were deposited into 96-well plates in limiting dilution and single colonies were injected into mice 14 days later. Lentiviral insertion site analysis was performed as described elsewhere [49] using the NlaIII restriction endonuclease. Supporting Information Figure S1 CoCSC phenotype cells preferentially survive CPA chemotherapy. UM-C6 tumors were initiated with ESA + CD44+ cells isolated by FACS. A) After randomization to normalize treatment groups at 400 mm3 at day 0, twice weekly administra- tion of vehicle versus 38 mg/kg CPA commenced and UM-C6 tumors were measured periodically. B) Representative phenotypic analysis of vehicle-treated control and CPA-treated tumors for human UM-C4 ESA+CD44+CD166+ cells. Mean6SEM. C) Representative overlay histogram displaying the CD166 expres- sion on human UM-C6 ESA + CD44+ cells. Found at: doi:10.1371/journal.pone.0002428.s001 (10.40 MB TIF) References 1. Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414: 105–111. 2. Clarke MF, Fuller M (2006) Stem cells and cancer: two faces of eve. Cell 124: 1111–1115. 3. Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, et al. (2004) Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast- crisis CML. N Engl J Med 351: 657–667. 4. 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