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

SC-12-0168_sm_SupplFigure1.pdf127KSupplementary Figure 1 Nuclear localisation of endogenous NANOG in CRC cells. Immunofluorescence staining for DAPI (blue), FLAG (green) and NANOG (red) indicates a nuclear localisation of endogenous NANOG in HCT116 cells. A) HCT116 cells transfected with FLAG-NANOG expression vector and stained with anti-FLAG (green arrowheads) to validate the anti-NANOG (red arrowheads) antibodies for IHC and IF staining. White arrowheads indicate DAPI:NANOG stained cells and yellow arrowheads indicate DAPI:FLAG:NANOG stained cells. B) Images indicate the endogenous NANOG protein expression (red arrowheads). White arrowheads indicate DAPI:NANOG stained cells. C) Images are the control cells stained for the secondary antibody only. Different CRC cell lines including HCT116 cells were used and in 2-3 independent experiments. D) IF staining for DAPI (blue) and NANOG (red) in ES cells transfected with NANOG-specific duplex siRNA or scrambled control siRNA.
SC-12-0168_sm_SupplFigure2.tif2002KSupplementary Figure 2 Differential expression of NANOG in different CRC cell lines and tissues. (A) Immunofluorescent staining for NANOG in mES cells (red) transfected with in pCMVNANOG- eGFP expression vector (green). (B) Fluorescent microscopic analysis to validate the NANOG1-promoter-eGFP construct in mouse embryonic stem (mES) cells that is used for CRC cell lines. Mouse embryonic fibroblast (MEF) used as feeder cells and showed no activity of NANOG1-promoetr. Bars: 200 μm. (C) Comparative expression of NANOG1- Luciferase reporter (NANOG1-Luc) constructs without (wt) and with mutations into indicated DNA-binding sites; M1 (M1mut), Oct (Octmut), Sox (Soxmut) and Oct - Sox together (Octmut/Soxmut) in mES cells. Bars: means ±s.e. from triplicate experiment (*P ≤ 0.05; **P ≤ 0.001, ***P ≤ 0.0001). (D) RT-PCR analyses of NANOG and HPRT genes. Primers (Table S1) that correspond to sequences of both NANOG1 and NANOGP8 were used. Purified PCR products were digested with the restriction enzyme AlwN1 and run on a 1% agarose gel and stained with Ethidium bromide (EtBr). A unique sequence for the restriction enzyme AlwN1 in NANOGP8 cDNA resulted 2-NANOGP8 cDNA fragments and NANOG1 remains uncut.
SC-12-0168_sm_SupplFigure3.tif665KSupplementary Figure 3 NANOG1-activity is positively regulated by β-catenin/TCF4 but not Gli1. (A, C and D) Comparative Luciferase expression of NANOG1-promoter reporter gene with and without mutations into M1, OCT, SOX- DNA binding sites in SW620, HCT116 and HEK293 cells were transfected by Gli1 (A), TCF4, β-catenin and −N-TCF4 plasmids (C), pSUPER-β-catenin-specific or -scrambled control siRNA (D). The luciferase activity of cells transfected with only NANOG1wt-Luc and tk-Rluc is arbitrarily set to 1. Bars mean ± s.e.m. in 3 independent experiments. (B) Western blotting analysis with FLAG, HA, c-JUN, TCF4, β- catenin and β-actin (loading control) antibodies for cell lysates of HCT116 cells that were transfected either with expression vectors for; pFLAG-cJun (left-top panel), pHA-TCF4 (leftmiddle panel) and pFLAG-β-cateninS33 (left-lower panel) or with specific duplex siRNA or scrambled control siRNAs for c-Jun (right-top panel), TCF4 (TCF7L2) (right-middle panel) and β-catenin (CTNNB1) (right-lower panel) genes respectively.
SC-12-0168_sm_SupplFigure4.pdf1682KSupplementary Figure 4 Over-expression of c-Jun induced tumour cell numbers in teratomas derived from ApcMin/+-ES cells. (A) IHC for anti-NANOG antibody indicate induction of Nanog expression (red vs. black arrowheads) in a proportion of intestinal tumour cells from ApcMin/+ mice. Figures are shown at low magnification (a) and at high magnification (b) Red arrowhead indicates NANOG stained and black arrowhead indicates unstained cells. Lower panel, (c) shows IHC for NANOG in representative sections of mouse normal intestinal tissue, hardly to see Nanog+ cells. Possible Nanog+ cells are shown with yellow arrowheads and at high magnification (d) Bars: (a, c) 50 μm; (b, d) 10 μm. (B) Western blotting analysis of c-Junwt and c-Jun-TA overexpression, stably transfected in ApcMin/+-ES cells (left panel). Asterisk (*) indicates a non-specific band. H&E stained sections of C57BL/6J -SCID mouse teratomas (middle panels) Bars: 100 μm. Teratomas derived from ApcMin/+ ES-cell lines with ectopic expression of c-Junwt, cJun-TA and plasmid control, injected subcutaneously into syngenic mice. Quantitative analysis of tumour cells in teratomas (right panel) was measured by comparing more homogenous and undifferentiated histological sections being taken 100 μm apart (n=10, 20x objective, *P ≤ 0.05) using AxioVision digital image processing software and normalised by number ApcMin/+-ES cells.
SC-12-0168_sm_SupplFigure5.pdf1669KSupplementary Figure 5 NANOG+ cells are co-localised with nuclear β-cateninHigh and low levels of MUC2 differentiation marker. (A) Immunofluorescence for endogenous β-catenin (b, green arrowheads) and endogenous NANOG (c, red arrowheads) indicates co-stained positive cells (d, yellow arrowheads). Coexpression and nuclear co-localization of β-catenin and NANOG positive cells showed with DAPI at (e, white arrowheads)-panel. Bars: 50 μm. (B) Immunofluorescence for endogenous β-catenin (b, red arrowheads) and Mucin2 (c, green arrowheads) indicates an inverse relationship between nuclear β-cateninHigh cells and MUC2 positive cells. These experiments were repeated at least 2-3 times in HCT116, DLD-1 and SW480 CRC cell lines. Bars: 50 μm. (C) Immunofluorescence for endogenous Mucin2 (b) and NANOG (c) in representative sections of HCT116GFP xenograft tumour sections. Clonogenic NANOG+ cells (c, yellow units) are stained with low intensity of DAPI (a, yellow units) and also expressing very low level of MUC2 (b, yellow units). Dashed white boxes show magnified single clonogenic unit. Bars: 50 μm.
SC-12-0168_sm_SupplFigure6.tif1160KSupplementary Figure 6 Over-expression of NANOG induced the cell division at G2/M. (A) Western blotting analysis using anti-GFP antibody of NANOG-GFP and GFP overexpression in HCT116 cells. (B) Western blotting for xenograft tumours derived from HCT116NANOG-GFP and HCT116GFP protein extracts with antibodies for NANOG, GFP and β- actin. (C) Graph shows percentage of Ki67+ cells by mean ± SEM, indicated by two independent researchers, n = 4; 8 images/tumour and for each cell line. (D) Graph shows number of NANOG+ cells by mean ± SEM; n=4 tumours; 12 colony units/tumour and for each cell line. (E) Schematic representative of cell cycle status for HCT116 cells with the GFP−NANOG and GFP overexpression, using Flow cytometry and PI staining analysis. (F) Representative genomic sequencing analysis of bisulfite converted products of the promoter region of NANOG gene methylation activity, showed differences between RFP+/GFP+ and RFP+/GFP− pre-sorted cells. hES cells were used as a control. (G) qRT-PCR analysis of NANOG1 mRNA in the RFP+/GFP+ and RFP+/GFP− pre-sorted cells. Results were normalized to HPRT expression in the same sample, and data are presented as fold over hES cells (mean ± SD; n = 3; **, P < 0.01; ***, P < 0.001). Experiment was performed in triplicate for each sample and repeated on at least three independent occasions. (H) RT-PCR and the restriction enzyme AlwN1 digestion analyses of NANOG and HPRT genes for the RFP+/GFP+ and RFP+/GFP− pre-sorted cells indicate a differential expression of NANOG cDNA expression. hES and hFib cells were used as controls.
SC-12-0168_sm_Supplinfor.pdf106KSupplementary Data
SC-12-0168_sm_Table.pdf24KSupplementary Table

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