Supplementary Information (Aoki, K. et al., "Chromosomal instability by β-catenin/tcf transcription in APC or β-catenin mutant cells")

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1 Supplementary Information (Aoki, K. et al., "Chromosomal instability by β-catenin/tcf transcription in APC or β-catenin mutant cells") Supplementary materials and methods ES cells and Mice The floxed β-catenin (i.e., Catnb +/lox(ex3) ) and β-catenin Δex3 (i.e., Catnb +/Δex3 ) ES cells were generated as described previously (Harada et al., 1999). The Apc / (i.e., Apc Δ716/Δ716 ) ES cells were isolated from Apc +/Δ716 ES cells (Oshima et al., 1995) by selection in 1 mg/ml geneticin (Sigma). The Apc Δ716, Catnb +/lox(ex3) :Krt1-19 +/cre and Catnb +/lox(ex3) :Tg Fabpl cre mouse strains were described previously (Harada et al., 1999; Oshima et al., 1995). All animal experiments were approved by the Animal Care and Use Committee of Kyoto University. Immunohistochemical Analyses Immunohistochemical procedures were described previously (Harada et al., 1999). Antibody for β-catenin was from Sigma, and antibody for p53 was from Oncogene Res. Prod. Determination of ABI Anaphase bridges were scored as described previously (Aoki et al., 2003), and tripolar cells were also included. Namely, for mouse tissues, 4 mice were analyzed for each genotype, and at least 100 anaphase cells per mouse were scored for the normal intestinal epithelium, and polyp adenomas, respectively. For cultured cells,

2 anaphase cells were scored from more than three independent microscopic fields. For human gastric cancer tissues, anaphase cells were scored from each tissue sample stained with H&E. A total of 55 intestinal-type gastric cancer samples, and 44 diffusetype (Mizoshita et al., 2003; Seno et al., 2002) were examined. Western Analyses and Cdc2 kinase assay Western blots and kinase assay were performed as described previously (Aoki et al., 2003). To determine the levels of Cdc2 phosphorylation, ES cells were treated with nocodazole at 0.2 µg/ml (sigma) or colcemid at 1 µg/ml (Sigma) and harvested at the indicated time points and processed for analysis (Figure 3d and e). For the Cdc2 kinase assay, ES cells were treated with either nocodazole or colcemid for 12 hours, and 200 µg of their lysates were used for each assay on histone H1 (CALBIOCHEM) as substrate. Bands were scanned and their intensities were determined using the NIHImageJ software. Antibodies for APC, cyclins B and D, Cdc2, c-myc, and p21 were from Santa Cruz Biotechnology. Antibody for cyclin E was from Pharmingen. Antibody for p-cdc2 (Y15) was from Cell Signaling Technology, and antibody for β-actin was from Sigma. Chromosomal Banding Analysis The ES cells were cultured in the presence of 0.1 µg/ml colcemid for 20 min and the chromosomes were analyzed using the GTW banding method (Hsieh, 1997). Twenty metaphases were examined from the mutants and wild type W3, and 30 metaphases were examined from the wild type W5. To avoid selection bias, the first 20 (for mutants and 2

3 W3) or 30 (for W5) analyzable metaphases encountered were included in the analysis regardless of the length of the chromosomes or differences in the quality of banding. Cells and Plasmids DLD-1 was described previously (Aoki et al., 2003). TCF4-VP16 was constructed from the full length human TCF4 and the activation domain of VP16, and was expressed using the TetOff system with 1 µg/ml doxycyclin (Clontech). Dominant-negative TCF1 and TCF4 constructs were kindly provided by Dr H Clevers, whereas TCF4ΔC was from Dr T Akiyama (Sekiya et al., 2004). The dn-tcf1 and 4, and TCF4ΔC plasmids were infected into the respective ES cell lines using the retrovirus expression vector pqcxip (Clontech). Transductant cells were selected by puromicin (Sigma) at 0.1 µg/ml for a month. Cell Cycle Analysis Cells cycle analysis was performed as described previously (Aoki et al., 2003). For the checkpoint analysis, ES cells were treated with nocodazole at 0.2 µg/ml or colcemid at 0.1 µg/ml and harvested at the time points indicated (Figure 3a and b). 10,000-50,000 of the cells were analyzed in FACScan (B&D). Data shown are representative sets from four repeated experiments. 3

4 Wnt Transcription Activity Transfection assays with β-catenin/tcf reporter plasmids (Upstate Biotechnology) were performed as described (Aoki et al., 2003). Data shown are a representative set of three experiments. TUNEL Assays ES cells were treated with nocodazole at 0.2 µg/ml for 24 hours and labeled with TdT, according to the manufacture's protocol (Wako, Japan). The experiments are repeated twice and scored for three independent fields. Legends to supplementary figures Supplementary Figure 1 Karyotype analysis of the wild-type and Wnt signal-activated (β-catenin Δex3 and Apc / ) ES cell lines. (a-d) Representative metaphase chromosomes of the wild-type (a), Apc / (b), and β-catenin Δex3 (c, d) ES cell subclones. Their karyotypes are shown in parentheses. Closed arrows in b indicate (13. 13) Robertsonian chromosomes. Closed and open arrows in c indicate additional chromosomes of chr 19 and chr 10, respectively. Closed arrows in d indicate (Y. Y) Robertsonian chromosomes. Supplementary Figure 2 Cell cycle analysis of the wild-type (WT) and Wnt signalactivated (β-catenin Δex3 and Apc / ) ES cell lines treated with colcemid or nocodazole. (a) Flow cytometric profiles of the wild-type (WT) and Wnt signal-activated (βcatenin Δex3 and Apc / ) ES cell lines treated with colcemid for the indicated durations. Boxed areas indicate the cells with DNA content > 8N. Numbers show mean ± s.d. of the 4

5 fraction/surviving cells from triplicate assays. (b) Polyploid (> 8N) cell fraction in surviving cells after treatment with colcemid for the indicated durations. * P < (c, d) Polyploid (> 4N) cell fraction in surviving cells after treatment with nocodazole (c) and colcemid (d) for the indicated durations, respectively. W, B and A indicate wild-type, β- catenin Δex3 and Apc / ES cells, respectively. * P < Supplementary Figure 3 Molecular analysis of the wild-type (WT) and Wnt signalactivated (β-catenin Δex3 and Apc / ) ES cell lines treated with nocodazole or colcemid. (a) Expression of cell cycle regulator proteins in the ES cell lines treated with nocodazole at the indicated time. β-actin was used as a loading control. (b) Fraction of Cdc2 phosphorylated at Y15 in total Cdc2, estimated from triplicate assays as shown in panel c. * P < (c) Molecular analysis of the cell cycle regulator proteins after colcemid treatment for the indicated durations. β-actin was used as a loading control. In panel a and c, W, B and A indicate wild-type, β-catenin Δex3 and Apc / ES cells, respectively. 5

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