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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.
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