Supplementary Information (Aoki, K. et al., "Chromosomal instabilityby 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-cateninΔex3 (i.e., Catnb+/Δex3) ES cells were

generated as described previously (Harada et al., 1999). The Apc–/– (i.e., ApcΔ716/Δ716) ES

cells were isolated from Apc+/Δ716 ES cells (Oshima et al., 1995) by selection in 1 mg/ml

geneticin (Sigma). The ApcΔ716, Catnb+/lox(ex3):Krt1-19+/cre and Catnb+/lox(ex3):Tg⋅Fabplcre

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, 300-700

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anaphase cells were scored from more than three independent microscopic fields. For

human gastric cancer tissues, 10-30 anaphase cells were scored from each tissue sample

stained with H&E. A total of 55 intestinal-type gastric cancer samples, and 44 diffuse-

type (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

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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.

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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-cateninΔex3 and Apc–/–) ES cell lines. (a-d) Representative metaphase chromosomes of

the wild-type (a), Apc–/– (b), and β-cateninΔex3 (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 signal-

activated (β-cateninΔex3 and Apc–/–) ES cell lines treated with colcemid or nocodazole.

(a) Flow cytometric profiles of the wild-type (WT) and Wnt signal-activated (β-

cateninΔex3 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

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fraction/surviving cells from triplicate assays. (b) Polyploid (> 8N) cell fraction in

surviving cells after treatment with colcemid for the indicated durations. *P < 0.01. (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-

cateninΔex3 and Apc–/– ES cells, respectively. *P < 0.01.

Supplementary Figure 3 Molecular analysis of the wild-type (WT) and Wnt signal-

activated (β-cateninΔex3 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 < 0.01. (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-cateninΔex3 and Apc–/– ES cells, respectively.