Supplemental material
Animal generation and treatments
The generation of Apc+/−, Apc−/−, RBP-J-/- and Atoh1-/-mice has been described elsewhere [1,
2, 3 , 4]. For the conditional invalidation of Apc and RBP-J, we crossed Apclox/loxVil-CreERT2
mice with RBP-Jlox/lox mice and obtained Apclox/loxRBP-Jlox/loxVil-CreERT2 mice. The
Apclox/loxVil-CreERT2 mice were also crossed with Atoh1lox/lox mice. All the mice analyzed
had a C57BL/6 background. Cre recombinase was activated by injecting tamoxifen (1 mg;
ICN) on four consecutive days. Four injections of tamoxifen induced the efficient
recombination of all floxed alleles (data not shown).
For proliferation studies, the mice were injected with BrdU (2.5 mg) (Sigma Aldrich) two
hours before they were killed. Duodenal segments were used for conditional gene targeting
studies.
Histology and immunohistochemistry
Immediately after the mouse was killed, its entire gastrointestinal tract was removed, splayed
open along its length and rolled up from the proximal to distal end to form a ‘Swiss roll’.
Tissues were then fixed by incubation in 4% formol overnight at 4°C and embedded in
paraffin wax. Hemalun/eosin and Alcian blue/Fast red staining was carried out on 3 µm
paraffin sections. For immunohistochemistry, 5 μm sections were treated with 3% hydrogen
peroxide for 15 minutes at room temperature. Antigen was retrieved by boiling in specific
buffer (see Supplementary Table 1) in a microwave pressure cooker (EZ retriever, Biogenex).
Sections were incubated in blocking solution (see Supplementary Table1 for details) for 20
minutes at room temperature. The sections were then incubated overnight at 4°C with primary
antibodies diluted in blocking solution. We used primary antibodies directed against β-catenin
(Transduction Laboratories; 1/50), Hes1 (generously provided by B.Z. Stanger, University of
Pennsylvania, Philadelphia, USA; 1/1000), BrdU (Abcam; 1/500), phosphorylated histone H3
(Upstate; 1/1000), Ki-67 (Novocastra; 1/300) and cleaved caspase-3 (Cell Signaling; 1/200),
Lysosyme (Dako; 1/500). Specific binding was detected with biotinylated secondary antibody
and ABC reagent (Vector) or with Envision-HRP (Dako). The signal was developed with
DAB (Vector). Apoptosis was analyzed by TUNEL, according to the kit manufacturer’s
instructions (Calbiochem). ImageJ software was used to count the cells positive for BrdU and
phosphorylated Histone H3 in each crypt. About 200 crypts were counted per mouse (3
animals per genotype).
In situ hybridization
Notch2 and Dll4 probes were produced by RT-PCR and inserted into the pGEM-T easy
vector (Promega, Madison, USA) and sequenced. Non-isotypic in situ hybridization was
carried out with DIG-labeled riboprobes prepared with a Roche DIG-labeled transcription kit
(Roche Molecular Biochemicals, Mannheim, Germany). Riboprobes were transcribed from
pGEM-T. Sections were deparaffinized, treated with 0.3x Triton in PBS, 0.1 mg/ml proteinase
K in 100mM Tris-EDTA. After washing in PBS, sections were treated with 0.25% acetic acid
containing 0.15 M triethanolamine and then prehybridized for two hours at 68°C in
hybridization buffer containing 1x salt solution (10x salt solution: 3 M NaCl, 100 mM Tris,
0.5 M NaH2PO4, 0.5 M Na2HPO4, 0.5M EDTA), 1x Denhardt’s solution, 10% dextran sulfate
and 0.1 mg/ml yeast rRNA (Sigma). Hybridization buffer was replaced by fresh buffer
containing 0.2 to 0.5 g/l DIG-labeled probes and the sections were hybridized overnight at
68°C. Slides were washed once for one minute in 5x SSC at 68°C, and for one hour at 68°C in
0.2x SSC. Signals were detected by immunochemistry, with alkaline phosphatase-conjugated
anti-digoxigenin Fab fragments (Roche Molecular Biochemicals, Mannheim, Germany) and
NBT/BCIP (Vector Laboratories, Burlingame, CA) as the substrate. Sections were
counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame,CA).
Cell culture, transfections and reagents
The colon carcinoma cell lines SW480, TC7 and HT29 were cultured in DMEM Glutamax
(4.5 g/l D-glucose with pyruvate) medium (Gibco) supplemented with 10% heat-inactivated
fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco), at 37°C, in a
humidified atmosphere containing 5% CO2.
For the siRNA assays, cells were transfected with the siRNA negative control and with two βcatenin siRNAs (10 nmol/l; Invitrogen), using Lipofectamine RNAiMAX as recommended by
the manufacturer (Invitrogen). For Atoh1 stability analysis, pCDNA-Atoh1-HA and ß-catenin
plasmid containing a deleted Ser-45 were transfected using Lipofectamine 2000 (Invitrogen).
Cells were harvested 48 hours after transfection. For Hes1 luciferase assays, after 24 hours in
culture (80% confluence), cells were cotransfected with 0.5 μg/ml of the indicated reporter
plasmid together with 0.5 μg/ml of the dominant-negative form of Tcf4 (ΔNTCF4), NICD or
empty vector plus 10 ng/ml of TK-Renilla reporter vector, in the presence of Lipofectamine
2000. The reporter plasmid, consisting of the firefly luciferase gene under the control of
various fragments of the Hes1 promoter (-2.5 kb, -0.4 kb or lacking the RBP-J sites), was
generously provided by R. Kageyama (Kyoto University, Japan) [5]. Reporter activity was
determined 48 hours after transfection, with the dual luciferase reporter assay system
(Promega). Relative luciferase activity was normalized against Renilla luciferase activity.
For Atoh1 and Muc2 luciferase assays, cells were cotransfected with 0.25 μg/ml of the
indicated pGL3 reporter plasmid, together with 5 ng/ml of the TK-renilla reporter vector, in
the presence of Lipofectamine 2000. The reporter plasmids were the firefly luciferase gene
under the control of the Atoh1enhancer-promoter regulatory sequences (Enh-Atoh1-Luc) [6],
or the MUC2 promoter covering the -371/-27 regions of MUC2 (Muc2-Luc) [7]. Twelve
hours after transfection, cells were treated with GSK3 inhibitor at a concentration of 12.5
µmol/l (AR-A014418, Sigma Aldrich) for 48 hours, or with proteasome inhibitor at a
concentration of 25 µmol/l (MG132, Calbiochem) for 18 hours. Luciferase activity was
determined with the dual luciferase reporter assay system (Promega). Relative luciferase
activity was normalized against Renilla luciferase activity.
Western blotting
Tissues were homogenized in Laemmli buffer (1:10 wt/vol). Total proteins (10 µg) were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and
were transferred to a nitrocellulose membrane and probed with antibodies against β-catenin
(Transduction Laboratories; 1/2000), Hes1 (a gift from T. Sudo, Toray Industries, Tokyo;
1/1000), NICD (Cell Signaling; 1/1000), HA (Roche, 1/500) and -actin (Cell Signaling;
1/1000). The signals were visualized with the chemiluminescence detection system.
RNA extraction and analyses
Total RNA from cells and tissues was isolated with TRIzol reagent (Invitrogen), according to
the manufacturer’s protocol. For RT-PCR, total RNA was reverse-transcribed with the
Transcriptor First-Strand cDNA Synthesis Kit (Roche Diagnostics). Quantitative RT-PCR
was carried out with a LightCycler Carousel-Based System and the LightCycler 480 System,
using the Light Cycler Fast Start DNA Master SYBR Green I Kit and the LightCycler 480
SYBR Green I Master Kit (Roche Diagnostics). Results are expressed relative to 18S rRNA.
PCR primer sequences are available from Supplemental Table 2.
Statistical analysis
We assessed the statistical significance of differences with nonparametric Wilcoxon matchedpair tests on 14 pairs of human colorectal carcinomas and normal tissues from matched
patients. The data presented are means ± SD for Hes1 mRNA levels and means ± SEM for the
fold-change in mRNA levels for Notch receptors and ligands. We used two-tailed Student’s t
tests for mouse samples and in vitro experiments. The data are expressed as means ± SEM.
Experiments were carried out on 10 normal tissues and 11 adenomas for the APC+/- mouse
model, three animals per genotype for conditional gene-targeted mice, three independent
cultures performed in duplicate for luciferase assays Statistical significance was as follows:
P< 0.001, extremely significant (***); 0.001 < P< 0.01, very significant (**); 0.01 < P< 0.05,
significant (*) and P>0.05, not significant (ns).
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