Supplementary Information
Experimental animals
All experiments were performed under the UK Home Office guidelines. Outbred male mice from
6–12 weeks of age were used, and they were segregating for the C57BLJ and S129 genomes.
The alleles used were as follows: Apc580S, Mycfl and AhCre (Ireland et al., 2004; Muncan et al.,
2006; Sansom et al., 2004). Cre activity was induced in control and experimental mice by 3
intraperitoneal injections of 80 mg/kg -naphthoflavone within 24 hours.
Intestinal Tissue Isolation
The proximal 7 cm was mounted en face, fixed overnight in methacarn (methanol, chloroform
and acetic acid; 4:2:1 ratio) and paraffin embedded. The following 5 cm were divided into 1-cm
lengths, bundled using surgical tape, and then fixed in 4% formaldehyde at 4°C for no more than
24 h before processing. The remainder was fixed in methacarn. Intestines from ageing AhCre+
Apcfl/+Mycfl/+ and AhCre+ Apcfl/+Myc+/+ mice were fixed in methacarn only.
Epithelial Cell Isolation
In order to obtain a population of cells enriched for epithelial cells, an epithelial extraction
protocol based on that of Bjerknes and Cheng (1981) was performed on freshly harvested
intestine. Briefly, a 10 cm section of small intestine (approximately 5-15 cm from the stomach)
was flushed well with water and PBS before being tied off at one end and inverted over a 4 mm
glass spiral. Vibration was then applied to the glass spiral and the intestine placed in 10 mM
EDTA in Hanks’ Balanced Salt Solution (HBSS; Gibco) at 37°C for 15 min. The intestine was
then moved into a clean tube of 10 mM EDTA/HBSS and incubated in the same fashion for a
further 15 min. Centrifugation (2700 x g, 4°C, 15 min) yielded a pellet containing predominantly
epithelial cells. The second extraction (15-30 min) was used for downstream qPCR analysis.
Histology and IHC
-catenin immunohistochemistry was performed on the intestines fixed in formalin as previously
described (Sansom et al., 2004). Crypt size and apoptosis were scored from H and E stained
sections as previously described (Sansom et al., 2007). For each analysis, 25 full crypts were
scored from at least 3 mice of each genotype. For BrdU labeling, mice were injected 2 h before
culling with 0.25 ml of BrdU (GE Healthcare UK Ltd, Little Chalfont, Buckinghamshire, UK).
The staining was performed using an anti-BrdU antibody (BD, Franklin Lakes, NJ) at 1 part in
100 on the intestines fixed in methacarn. At least three mice were used for each genotype.
RNA extraction
RNA was extracted from tissue enriched in epithelial cells using the RNeasy Mini Kit (Qiagen,
Crawly, West Sussex, UK) according to the manufacturer’s instructions. DNA-free
(Ambion/Applied Biosystems, Warrington, UK) was used to treat the RNA prior to RT-PCR to
eliminate genomic DNA contamination, according to the manufacturer’s instructions (rigorous
treatment protocol).
Quantitative PCR (qPCR) and Data Analysis
Total RNA (3 g) was reverse transcribed to cDNA using the Superscript III kit (Invitrogen,
Paisley, UK) according to the manufacturer’s instructions. Quantitative PCR was performed
using the Chromo4 system (Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, UK) and
DyNAmo HS SYBR Green qPCR kit (Finnzymes, Espoo, Finland). PCR was performed in
triplicate in a 20 l reaction mixture containing 10 l of 2 × DyNAmo HS master mix, 1 l of
each of the primers (final concentration 0.5 M), and 1 l cDNA. The reaction mixture without a
template was run as a control. The cycling conditions were as follows: 95°C for 15 min,
followed by 40 cycles of three steps consisting of denaturation at 94°C for 15 s, primer annealing
at the optimal temperature for 30s, and primer extension at 72°C for 30s. A melting curve
analysis was performed from 70°C to 95°C in 0.3°C intervals to demonstrate the specificity of
each amplicon and to identify the formation of primer dimers.-actin and cytochrome c-1 were
used to normalize for differences in RNA input. Reference genes were selected from a panel of
eight
genes
using
the
geNorm
software
(http://medgen.ugent.be/~jvdesomp/genorm/)
(Vandesompele et al., 2002). The calculation of relative expression ratios was carried out with
the Relative Expression Software Tool (REST) 2008 (http://www.gene-quantification.com/)
using the pairwise fixed randomization test (Pfaffl et al., 2002).
Primers
Gene
Primer set (5’-3’)
Actb
-
60
Cyc1
-
60
Ascl2
Axin2
Ccnd2
Cd44
Cdk4
Ephb3
Lgr5
Myc
Nkd1
Tcf7
Tiam1
Tnfrsf12a
Tnfrsf19
GAGCAGGAGCTGCTTGACTT
TCCGGAAGATGGAAGATGTC
GCTCCAGAAGATCACAAAGAGC
AGCTTTGAGCCTTCAGCATC
CTACCGACTTCAAGTTTGCC
GCTTTGAGACAATCCACATCAG
GCCTCAACTGTGCACTCAAA
GTGTTTCAGGGGTGGTCATC
AATGTTGTACGGCTGATGGA
AGAAACTGACGCATTAGATCCT
CGTGAAAGTGGACACCATTG
CCAAGTAGAAGCCAGCCTTG
GAGTCAACCCAAGCCTTAGTATCC
CATGGGACAAATGCAACTGAAG
TGAAGAAGAGCAAGAAGATGAG
CTGGATAGTCCTTCCTTGTG
TTTCAACCATACCGATCTGC
CTCCTCTCAATGTTCTCATCC
CACCACAGGAGGAAAAAGAAATG
CAAGGAGGGCAACAGAAGATAC
TGCAGTTCCCCAGAGAGC
CACACAATCTCTTGCCTCCA
CTGGTTTCTAGTTTCCTGGT
CTTGTGGTTGAAAGAGTCTG
CTGGATTTGAAGTTTGTCTG
CGTGTTTATTCCTGCTACTC
Annealing
temp. (oC)
Notes
Part of the geNorm kit from
PrimerDesign Ltd
Part of the geNorm kit from
PrimerDesign Ltd
60
62
60
62
60
61
58
58
60
60
Designed by PrimerDesign
Ltd
62
60
55
Supplementary references
Ireland H, Kemp R, Houghton C, Howard L, Clarke AR, Sansom OJ et al (2004). Inducible Cremediated control of gene expression in the murine gastrointestinal tract: effect of loss of betacatenin. Gastroenterology 126: 1236-46.
Muncan V, Sansom OJ, Tertoolen L, Phesse TJ, Begthel H, Sancho E et al (2006). Rapid loss of
intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cell Biol.
Pfaffl MW, Horgan GW, Dempfle L (2002). Relative expression software tool (REST) for
group-wise comparison and statistical analysis of relative expression results in real-time PCR.
Nucleic Acids Res 30: e36.
Sansom OJ, Meniel VS, Muncan V, Phesse TJ, Wilkins JA, Reed KR et al (2007). Myc deletion
rescues Apc deficiency in the small intestine. Nature 446: 676-9.
Sansom OJ, Reed KR, Hayes AJ, Ireland H, Brinkmann H, Newton IP et al (2004). Loss of Apc
in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev 18: 138590.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A et al (2002). Accurate
normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal
control genes. Genome Biol 3: RESEARCH0034.