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SC_12-0063_sm_supplFigure1.tif561KSupplementary Figure 1. Distribution of ALDH activity in one CCIC line (AG2) stained with PKH26 and, after 10 days, separated in three PKH-retaining fractions and assayed with the ALDEFLUOR kit as described in Materials and Methods. Percentages of ALDH-positive cells, deducted of the respective DEAB (control) samples, are 5.5 (PKHLOW/NEG), 5.2 (PKHMED) and 5.7 (PKHHIGH).
SC_12-0063_sm_supplFigure2.pdf1502KSupplementary Figure 2. Immunofluorescence staining of CD133 (top panels) and Lgr5 (bottom panels) expression in seven lines of CCIC (STEM) and their differentiated progeny (DIFF). 60x magnification, 2x zoom. Abbreviations: Lgr5, leucine-rich repeat containing G protein-coupled receptor 5.
SC_12-0063_sm_supplFigure3.pdf1150KSupplementary Figure 3. (A): Immunofluorescence analysis of Plk1 localization and phosphohistone H3 (pH3) expression in one CCIC line (CRO stem), its differentiated progeny (CRO Diff) and HeLa cells. 60x magnification, 3x zoom. (B): Screening of a kinase inhibitor library and chemotherapeutic agents on one CCIC line (CCIC CRO). CCIC were treated for 48 hours with the indicated kinase inhibitors and chemotherapeutic agents and cell viability was measured with the Cell Titer Glo assay as described in the Materials and Methods section. The black arrow indicates the BI 2536-treated sample. (C): Dose-response treatment of a CCIC line (CCIC CRO) with BI 2536 (BI) at the indicated times. Cell viability was measured as described in the Materials and Methods section. Abbreviations: CCIC, colon cancer-initiating cells; Plk1, Polo-like kinase1; pH3, phospho Histone H3.
SC_12-0063_sm_supplFigure4.pdf952KSupplementary Figure 4. (A): Proliferative activity of colon cancer stem (Stem) versus differentiated (Diff, CCIC-derived) cells. 2,5 x 104 cells were plated in 12-well plates and proliferation was monitored by manual counting. Bars represent the fold increase in cell number assessed 10 days after plating. (B): BrDU staining of CCIC (AG2 stem) and of the same cells differentiated for 10 days in serum-containing medium (AG2 Diff) as described in Materials and Methods. (C): Double Ki67/CD133 staining of an untreated CCIC (AG2)-derived xenograft.
SC_12-0063_sm_supplFigure5.pdf1184KSupplementary Figure 5. (A): Histone H2AX phosphorylation (pH2AX) in CCIC AG2 untreated (-) or treated for 24 hours with 100 nM BI 2536 (BI 2536) detected by immunofluorescence (60x magnification, 2x zoom). (B): TMRM staining and flow cytometry analysis of CCIC untreated (CTRL) and treated for 72 hours with 100 nM BI 2536. Numbers indicate the percentage of cells with depolarized (TMRM-negative) mitochondria contained in the left inset. (C): Cell death in two CCIC lines (1.1 and DN08) untreated or treated 48 hours with 100 nM BI 2536 in the presence or in the absence of zVAD-FMK. Death induced in Jurkat cells by TRAIL in the presence or in the absence of zVAD-FMK is shown as a control of zVAD-FMK efficacy. Data shown are the mean ± SD of three independent experiments. (D): Photomicrograph showing the size of CCIC AG2 untreated (-) or treated for 48 hours with BI 2536 (BI 2536) and stained with Dapi and Alexa488- conjugated phalloidin (60x magnification, 3x zoom). Abbreviations: CCIC, colon cancer-initiating cells; Plk1, Polo-like kinase1; pH2AX, phospho Histone H2AX; zVAD, benzyloxycarbonyl-Val- Ala-Asp (OMe) fluoromethylketone; TRAIL, TNF-related apoptosis-inducing ligand.
SC_12-0063_sm_supplFigure6.pdf1515KSupplementary Figure 6. (A): Immunohistochemistry staining for cleaved cytokeratin 18 (CK18) on CCIC (AG2)-derived xenograft sections after 48 hrs of mice treatment with vehicle (-) or with BI 6727 (BI 6727), 20X magnification. (B): Immunofluorescence staining (40x magnification) for Lgr5 and TUNEL on CCIC (AG2)-derived xenograft sections derived from mice treated for 48 hrs with vehicle (Vehicle) or with BI 6727 (BI 6727). Abbreviations: CK18, cytokeratin 18; Lgr5, leucine-rich repeat containing G protein-coupled receptor 5
SC_12-0063_sm_supplTable1.pdf145KSupplementary Table 1. Percentages of tumor incidence in NSG mice inoculated with the indicated number of cells of three different CCIC lines (DN08, AG2, 18). Cells were stained with PKH26, sorted according to PKH26 retention 7-10 days after staining and immediately inoculated in NSG mice (2 for control groups and 4 for PKH groups). Tumor incidence was evaluated approximately two months after injection.
SC_12-0063_sm_supplTable2.pdf327KSupplementary Table 2. Tumor type, Dukes' classification and staging of colorectal tumors that were used to derive the indicated CCIC lines. The mutational status of p53, KRAS (G12V and G13D) and PI3 kinase (PI3CA) of the respective CCIC lines is shown (wt, wild type; mut, mutated; fs, frameshift).
SC_12-0063_sm_supplTable3.pdf72KSupplementary Table 3. Compounds included in the screening of potentially cytotoxic agents on CCIC, as shown in Figs. 2C and Supplementary 3B.

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