Supporting Material and Methods
DEN-mouse model
For our experiments we used male C3H/He mice because of their susceptibility to liver cancer
(1, 2). Mice were obtained from Harlan Winkelmann GmbH, Germany. The induction of liver
tumors in male C3H/He mice was initiated at age 6 weeks by a single i.p. injection of DEN
(90 µg/kg). Mice were fed with a PB (0.07% w/w) containing standard diet, starting 2 weeks
after DEN intoxication. Animals were sacrificed and tumors prepared at week 32, 37, 42, and
56. Control animals were fed only with standard diet (ssniff PS M-Z obtained from ssniff
Spezialdiäten GmbH, Germany) and sacrificed at different points in time (11, 16 and 42
weeks). The study protocol was approved by the public health department of Austria
(approval No: GZ 66.010 – 27).
Pathohistological examination and micro-dissection
Liver and tumor tissues were either snap frozen and stored in liquid nitrogen or fixed in
formaldehyde (8% buffered formaldehyde solution) and embedded in paraffin. Dewaxed
sections (3 m) were stained with hematoxylin and eosin (H&E) following standard
procedures and evaluated by light microscopy by a pathologist (C.L.) for the presence of liver
tumors. The tumors were classified into hepatocellular neoplasias resembling adenomas or
carcinomas based on published criteria (3).
Cryosections were stained with H&E and examined for the presence and location of lesions.
Tumor as well as normal tissue samples were collected from hematoxylin stained parallel
sections by laser microdissection using the PALM Laser Micro-dissection and Pressure
Catapulting (LMPC) system (Zeiss, Vienna, Austria) according to previously published
protocols (4).
Mouse whole genome amplification
The laser micro-dissected lesions frequently consisted of approximately 500-1.000 cells. The
obtained DNA was not sufficient for subsequent analyses. Therefore, we subjected the
extracted DNA to unbiased whole genome amplification employing the GenomePlex Single
Cell WGA-Kit (#WGA-4, Sigma-Aldrich, Vienna, Austria) as described previously (4, 5).
Quality control of the amplified DNA was performed by multiplex PCR according to a
protocol for human DNA (6), which we had adapted for mice samples (5).
Array-CGH
Test DNA and reference mouse DNA were labeled with different fluorescent dyes (Cy3 and
Cy5, respectively) and co-hybridized on 4x44K Agilent mouse arrays. The reference DNA,
derived from the same mouse strain, i.e. C3H/He (JaxLab #000659), was also amplified using
the GenomePlex Single Cell WGA-Kit. In all array-CGH analyses sex mismatched reference
DNA was used, i.e. the tumor DNA was always derived from male animals, whereas the
reference DNA was always female. The feature extraction program calculates log2 ratios
between green (Cy3) and red (Cy5) fluorescence. Subsequently, the Genomic workbench
5.0.14 program was used for analysis and visual representation of gains and losses
(amplifications and deletions) in the test DNA (for details see 5). The Genome workbench
5.0.14 settings were as follows: aberration algorithm ADM-2, threshold 6.0, Fuzzy Zero:
OFF, aberration filter: minimum number of probes = 5 and minimum average absolute log2
ratio = 0.27, corresponding to a fold change of 1.21.
Evaluation by GISTIC (Genomic Identification of Significant Targets in Cancer)
GISTIC is designed for analyzing chromosomal aberrations in cancer (7). GISTIC calculates
statistical significance of copy number aberrations obtained by array-CGH. GISTIC results
are represented graphically by plots and also as text files containing the lists of significantly
deleted and amplified genes. As the original program had only existed for human array-CGH
files, we adapted it for Agilent text input files and for the mouse genome.
Mutation screening -catenin and H-Ras
We analyzed exon 3 in the β-catenin gene and codon 61 in the H-Ras gene by traditional
capillary sequencing. The same amplified DNA which we had applied for array-CGH was
used for this analysis.
Immunohistochemistry for -catenin and Ki67
We performed immunohistochemistry on formalin fixed paraffin embedded liver sections
derived from 32 and 56 week-old DEN-treated mice with antibodies directed against βcatenin (rabbit-monoclonal, dilution 1:100; Abcam, Cat. No: ab32572) and the nuclearprotein
Ki67 (rabbit-monoclonal, dilution 1:200; Abcam, Cat. No: ab15580) as a proliferation marker.
For both IHC staining experiments the slides were incubated with the primary antibody for 1
hour at room temperature (RT). As secondary antibody we applied Envision+System HRP
labelled polymer (anti-rabbit; Dako, Cat. No: 400211) for 30 min at RT. Detection was done
with DAB for 10min (β -catenin staining) and 5 min (Ki67 staining).
The areas of highest density β-catenin and Ki67-positive nuclei were selected and positive
nuclei were counted in 3 High Power Field (Olympus BH-2, field diameter 0,625 mm) in
tumors and non-neoplastic liver tissue.
For the statistical analysis we used Kruskal Wallis Test and Mann Withney U Test.
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