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Supplementary Materials
Materials and Methods
Cell line, transfection procedure, netrin-1 purification and soft agar assays :
Transient transfections of HEK293T cells were performed using lipofectamine reagent
(Invitrogen). Netrin-1 was purified from netrin-1 producing 293-EBNA cells according to
Serafini et al. 23. Soft agar assays were performed as described previously 21.
Plasmids constructs :
Full-length DCC expressing construct pDCC-CMV.S and netrin-1 expressing construct
pGNET1-myc were described previously 3. pTS4 Fabpl
(Univ. St Louis, Washington). The Fabpl
4x at –132
4x at –132
was from J.I. Gordon
promoter cassette was amplified by
PCR using the respective primers pTS4FORBglII 5’- TAT AGA TCT TCA GAA TAC
AAA ACA GCT TTA GGG-3’ and pTS4REVHindIII 5’-TAT AAG CTT CCC TGA CCA
CAA CAG CTC TGT CTG-3’ containing respectively a Bgl II and a Hind III sites and
inserted in Bgl II/Hind III of pcDNA3.1. This promoter corresponds to nucleotides –596
to +21 of the Fabpl gene, with the multiple insertion of a 35-base element at –132 that
leads to the expression of the transgene in the gut (and to a lesser extent in the
kidneys) of adult mice
18.
The construct used for transgenesis pcDNA-Fabpl-netrin-1
was finally obtained by inserting the full-length chick netrin-1 cDNA (EcoRI-XhoI) into
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pcDNA 3.1-Fabpl.
In Situ Hybridization :
DIG-labelled sense and antisense RNA probes corresponding to chick netrin-1 were
generated according to the methods described in the Roche "DIG RNA labelling kit". In
situ hybridization was performed on 18-µm-thick coronal cryostat sections through the
colon. Briefly, slides were successively treated with 10 mg/ml proteinase K (Roche) 5
mn at 37°C, and with acetic anhydride in 0.1 M triethanolamine for 10 min. They were
then dehydrated through graded ethanol solutions and hybridization was performed at
56°C for 15 h. Slides were washed in SSC at 56°C, followed by a formamide wash at
56°C and SSC washes at 37°C. DIG-labelled RNA hybrids were reacted with alkaline
phosphatase-conjugated anti-DIG antibodies (Roche). Reaction product was visualized
by incubating the sections with nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3indolylphosphate (Roche) overnight at room temperature.
Immunochemistry:
Immunohistochemistries on 4-µm-thick sections of colon or small intestine were
performed using formalin-fixed, paraffin-embedded tissue and high temperature antigen
retrieval treatment (DakoCytomation). Anti-UNC5H2 and UNCH3 (R&D systems), antinetrin-1 (Ab-2) (Oncogene), anti-DCC (A-20) (Santa Cruz), anti- Catenin (clone 14)
(Transduction Laboratories), anti-c-myc (9E10) (Roche), and anti-BrdU (Valbiotech)
antibodies were used. In the case of UNC5H3 and UNC5H2, recombinant ectodomains
of these proteins were used as competitive peptides (R&D systems). In the case of
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DCC, a competitive peptide (SantaCruz) was used.
RT-PCR:
Total RNA was isolated using Nucleospin RNAII kit (Macherey Nagel) from different
tissues. cDNA was prepared using random hexamers and Superscript TMII RnaseHreverse transcriptase (Invitrogen) according to the manufacturer’s protocol. For PCR,
(Invitrogen) according to the manufacturer’s instructions. The following mouse primers
were used for PCR: Netrin-1 forward :5’-TGACTGTAGGCACAACACGG-3’ , Netrin1reverse :5’-GGCATTACCCAACAGCAAGT-3’ ; -actin forward :5’CGGCCAGGTCATCACTATTG-3’ , -actin reverse :5’-CTCAGTAACAGTCCGCCTAG
-3’ .
To assay netrin-1/DCC expression in human colorectal tumors, total RNA was extracted
from biopsies of patients undergoing surgery for colorectal cancer using Nucleospin
RNAII kit (Macherey-Nagel) and 1 g was reverse-transcribed using Super-Script II
Reverse Transcriptase Rnase H-(Invitrogen).
Real-time quantitative RT-PCR was performed on ABI Prism 7700 (Roche) using the
Light Cycler FastStart DNA Master SYBERGreen I kit (Roche). Reaction conditions for
all optimal amplification as well as primers selection of both netrin-1 and DCC were
determined as already described
24.
The ubiquitously expressed RxR and GAPDH
genes showing the less variability in their expression between normal and colorectal
tumoral tissues were used as internal controls .
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Intestinal villi preparation and immunoprecipitation of tagged netrin-1:
Villi and crypts were isolated from the small intestine by mechanical dissociation using a
cold
solution
containing
2.5
mM
EDTA
and
250
mM
NaCl
(pH
7.5).
Immunoprecipitations and western-blot were carried out on wild type or transgenic
villi/crypts preparation using anti-c-myc antibody as previously described 25.
Mice generation and genotyping :
A linear and purified Avr II- SspI DNA fragment of the pcDNA-Fabpl-netrin-1 construct
was then microinjected into the male fertilized pronucleus obtained from C57BL/6 mice.
Mice founders were obtained by SEAT transgenesis center (CNRS - Villejuif, France).
Germline transmission and genotyping were detected by Southern-blot analysis of tail
genomic DNA. The various lines studied of tg-netrin-1 mice developed and bred
normally without any obvious gastrointestinal defects. In one line of transgenic mice,
large cystic malformative lesions with hamartomatous components were observed in
one or both kidneys in 90% of the animals. However, because this defect appeared in
only one line of mice, we cannot exclude the possibility that this defect is due to
transgene insertion, rather than to a specific netrin-1 effect. APC
+/1638N
mice were
obtained from R. Fodde and mated with parental or tg-netrin-1 mice. APC 1638N
mutation was detected by PCR as described before
26.
All animals have been used in
C57BL/6 genetic background.
Tumor analysis :
Transgenic and wild type animals were killed at 20 months. Liver and lungs of all
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animals were examined for the presence of metastases. The entire intestine was
removed, identical portions of colon and small intestine were resected, formalin fixed
and paraffin embedded. 4 µm-thick sections were prepared and stained with
hematoxylin-eosin-saffron. Histological examination and grading was conducted in a
single blinded fashion.
APC +/1638N and tg-netrin-1 APC +/1638N mice were killed at 6 months. The entire intestine
was removed and opened longitudinally; adenomas were resected, formalin-fixed and
paraffin embedded. 4 µm-thick sections were prepared and stained with hematoxylineosin-saffron. Histological classification and grading of neoplastic lesions was
performed in a blinded fashion and according to standard procedures.
Apoptosis scoring:
4 µm-thick tissue sections prepared from colon specimens and stained with
hematoxylin-eosin-saffron were used. Apoptotic epithelial cells were identified on
morphological
criteria,
including
evidence
of
nuclear
(pyknosis,
karyorrhexis,
fragmentation) and cytoplasmic (shrinkage, increased eosinophilia, detachment from
adjacent cells) alterations. The number of apoptotic cells was counted at a
magnification of x400, in at least 100 consecutive crypts and the intervening surface
epithelium.
23. Serafini, T. et al. The netrins define a family of axon outgrowth-promoting proteins
homologous to C. elegans UNC-6. Cell 78, 409–424 (1994).
24. Latil, A. et al. Quantification of expression of netrins, slits and their receptors in human
prostate tumors. Int. J. Cancer 103, 306–315 (2003).
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25. Forcet, C. et al. The dependence receptor DCC (deleted in colorectal cancer) defines an
alternative mechanism for caspase activation. Proc. Natl Acad. Sci. USA 98, 3416–3421
(2001).
26. Smits, R. et al. E-cadherin and adenomatous polyposis coli mutations are synergistic in
intestinal tumor initiation in mice. Gastroenterology 119, 1045–1053 (2000).
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Supplementary Figure Legends
Supplementary Figure 1. DCC inhibits anchorage-independent growth through
apoptosis regulation.
293T cells were transiently transfected with either a mock plasmid or with DCCexpressing construct in the presence or absence of netrin-1 3 and were allowed to grow
for 10.5 days in soft agar.
previously described
3
DCC expression was monitored by Western blot as
(not shown). The caspase inhibitor zVAD.fmk was also used
where indicated at 20M, added repeatedly every two days. (a) A set of representative
plates is shown. (b) The colony number was determined by counting isolated clones
with a diameter larger than 100m. Standard deviations are indicated (n=3).
Supplementary Figure 2. Netrin-1 overexpression does not modulate proliferation and
differentiation of the intestinal epithelium.
1 mg of BrdU was injected in either parental (a,c) or tg-netrin-1 (b,d) mice and animals
were sacrified 2 hours later. Anti-BrdU staining was then performed as described in the
method section on either small intestine (a,b) or colon (c,d). No significant difference
was observed between the five lines of transgenic mice tested and the parental mice.
Goblet specific staining was performed using Alcian blue staining on either parental
(e,g) or tg-netrin-1 (f,h) mice. (e,f) small intestine, (g,h) colon. Original magnifications:
a-b, x100, c-g: 120.
Supplementary Figure 3. Increased cell death in the gastrointestinal tract of netrin-1
mutant mice.
Representative microscopic images from hematoxylin-eosin-saffron staining of newborn
netrin-1 mutant mouse (a) or parental mouse (b). Arrows indicate cells with apoptotic
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morphology. Original magnifications a-b, x450. Quantitative analysis of apoptotic cells
in either wild-type or netrin-1 mutant mice performed as in Figure 2 is given in the main
text. The netrin-1 mutant mice were described previously 4. Small intestine and large
intestine from 3 parental and 3 netrin-1 -/- newborn mice have been analyzed
(p<0.0001).
Supplementary Figure 4. Netrin-1 and netrin-1 receptors localization in the intestinal
epithelium and in hyperplastic/neoplastic lesions.
Immunohistochemistry of UNC5H2 (a,b) and UNC5H3 (c,d) on small intestine section.
(b,d) competitive proteins. Immunohistochemistry of tagged netrin-1 (e-g) or DCC (h) in
tg-netrin-1 mice: (e) hyperplasia, (f,h) adenoma, (g) adeno-carcinoma. N: normal
epithelium, * Adenoma. Original magnifications: a-d, x100, e, x80, f, x120, g, x200, h,
x120.
Supplementary Figure 5. Netrin-1 is overexpressed in 7% of human colorectal tumors.
Expression analysis of Netrin-1 and DCC was performed on either 43 colorectal tumors
or adjacent normal tissues by RT-PCR. In 3 cases shown here, netrin-1 mRNA was
found overexpressed in the tumor (T) compared to the normal tissue (N) (a-b), (a)
quantitative PCR. The expression level of netrin-1 is given as the ratio between netrin-1
and GAPDH fluorescent signals (b) PCR in the exponential phase. Interestingly, while
DCC expression was found reduced by at least 3 fold in 70 % of the tested tumors (not
shown), in the 3 cases shown in (a-b) DCC expression was either maintained or
increased (b).
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