Stem Cell Cd44v Isoforms Promote Intestinal Cancer Formation in Apc(Min) Mice Downstream
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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