SupplementaryInformation

Stem cell CD44v isoforms promote intestinal cancer formation in Apc(min) mice downstream of Wnt signaling

Jurrit Zeilstra, Sander P.J. Joosten, Harmen van Andel, Cornelia Tolg, Anton Berns, Margriet Snoek, Marc van de Wetering, Marcel Spaargaren, Hans Clevers and Steven T. Pals

Figure S1

Laser-capture microdissection of epithelial cells from the colon of familial adenomatous polyposis patients. (A and C) Representative examples of normal crypts and of an early neoplastic lesion. (B and D) The same sections with the microdissected area removed.Snap-Frozen colonic tissue of three FAP patients wascut into 10-µm-thick sections, which were mounted on polyethylene covered glass slides (PALM Technologies, Bernreid, Germany).Sections were stained with hematoxylin for 1 minute and epithelial cells were dissected using a Veritas Microdissection System (Molecular Devices Corporation, CA, USA). RNA was isolated using the PicoPure RNA Isolation Kit (Molecular Devices Corporation) according to the manufacturer’s protocol.

Figure S2

Assessment of proliferation and apoptosis in the intestines of Cd44 variantmice.(A) Anti-Ki67 staining (SP6; Lab Vision, CA, USA)of an intestinal cross-section of a Cd44v4-10/v4-10mouse. Cycling CBC cells are indicated by dashed lines and cycling TA cells are indicated by arrows. (B) Intestinal cross-section stained using antibody that detects activated (cleaved) caspase-3 (Asp175; Cell Signaling Technology, Inc, MA, USA). An apoptotic epithelial cell in the stem cell compartment is indicated (arrow). (C) Quantification of the number of cycling (Ki-67 positive) CBC cells in the intestinal stem cell compartments of Cd44+/+, Cd44-/-, Cd44s/s, and Cd44v4-10/v4-10 mice. Samples were developedusing DAB(Sigma-Aldrich, Zwijndrecht, The Netherlands)and counterstained with hematoxylin.(D) Quantification of the number of cleaved-caspase-3 positive epithelial cells in the intestinal stem cell compartments. (E) Quantification of the number of Ki67-positive TA cells. (F) Crypt/villus length ratio’s (columns, mean; bars, s.e.m.; n=8 per group; ns, not significant; scale bar = 50 µm).

Figure S3

Assessmentof proliferation and apoptosis in the intestines of Cd44 variant ApcMin/+ mice.(A) Quantification of the number of cycling (Ki-67 positive) CBC cells in the intestinal stem cell compartments of Cd44+/+/ApcMin/+, Cd44-/-/ApcMin/+,Cd44s/s/ApcMin/+, and Cd44v4-10/v4-10/ApcMin/+mice. (B) Quantification of the number of cleaved-caspase-3 positive epithelial cells in the intestinal stem cell compartments. (C) Quantification of the number of Ki67-positive TA cells. (D) Crypt/villus length ratio’s (columns, mean; bars, s.e.m.; n=8 per group; *** P0.001; ns, not significant).

Table S1. PCR oligonucleotides. (A) Exon specific RT-PCR. (B) qRT-PCR.

(A) Exon specific RT-PCR.

Mouse

TargetOrientationSequence (5’ 3’)

Exon-3seTTCCGAGGATTCATCCCAAC

Exon-16 asCCTTGGATGAGTCTCGATCTC

Exon-5seAGAAGAGCACCCCAGAAAGC

Exon-v1 seAAGCCATGCAGCAGCTCAG

Exon-v2seACACCACCCAAGAGGCAAG

Exon-v3seGCTGGGAGCCAAATGAGG

Exon-v4seTTCTGCCCGCACAGAAGAC

Exon-v5seGACAGAATCAGCACCAGTGC

Exon-v6seAGTACAGCAGAAGCAGCAGC

Exon-v7seCCACAACAACCATCCAAGTC

Exon-v8seGACTCCAGTCATAGTACAACCCT

Exon-v9seTCTCTACATTACATGGAGAGCC

Exon-v10seCGGCGCTAAAGATGCAAG

Human

TargetOrientationSequence (5’ 3’)

Exon-2seGATGGAGAAAGCTCTGAGCATC

Exon-16 asTTTGCTCCACCTTCTTGACTCC

Exon-5seAAGACATCTACCCCAGCAAC

Exon-v2 seGATGAGCACTAGTGCTACAG

Exon-v3aseACGTCTTCAAATACCATCTC

Exon-v3bseTGGGAGCCAAATGAAGAAAA

Exon-v4seTCAACCACACCACGGGCTTT

Exon-v5seGTAGACAGAAATGGCACCAC

Exon-v6seCAGGCAACTCCTAGTAGTAC

Exon-v7seCAGCCTCAGCTCATACCAGC

Exon-v8seTCCAGTCATAGTATAACGCT

Exon-v9seCAGAGCTTCTCTACATCACA

Exon-v10seGGTGGAAGAAGAGACCCAAA

(B) qRT-PCR

Mouse

Cd44v6seCCTTGGCCACCACTCCTAATAG

asCAGTTGTCCCTTCTGTCACATG

Cd44v4seCCTTGGCCACCATTGCAAG

asCAGCCATCCTGGTGGTTGTC

total Cd44seGCACTGTGACTCATGGATCC

asTTCTGGAATCTGAGGTCTCC

Olfm4seGCCACTTTCCAATTTCAC

asGAGCCTCTTCTCATACAC

Ascl2seCTACTCGTCGGAGGAAAG

asACTAGACAGCATGGGTAAG

ActinseGGATGCAGAAGGAGATTACTG

asCCGATCCACACAGAGTACTTG

Human

CD44c5seAGTGAAAGGAGCAGCACTTCA

CD44c16-c5asGGTCTCTGGTAGCAGGGATTC

CD44v6-c5asGCCTGGATGGTAGCAGGGATTC

CD44v4-c5asGGTTGAAATGGTAGCAGGGATTC

Table S2. PCR oligonucleotides used for genotyping CD44 variant mice.

TargetOrientationSequence (5'  3')

hyg-rseCCTGATGCAGCTCTCGGA

asGGCCATTGTCCGTCAGGA

Cd44c3seGTATGGGTTCATAGAAGGAAATG

Cd44c20asGCCACCGTTGATCACCAGC

Cd44v4asCAGCCATCCTGGTGGTTGTC

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