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MS #ONC-2015-01780, REVISED2
SUPPLEMENTARY INFORMATION
Intestinal knockout of Nedd4 enhances growth of Apcmin tumors
Chen Lu1, Cornelia Thoeni1, Ashton Connor2, Hiroshi Kawabe3, Steven Gallinger2, Daniela
Rotin1,*
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Cell Biology Program, The Hospital for Sick Children, 686 Bay St, and University of Toronto,
Toronto, Ont, Canada M5G 0A4; 2Mount Sinai Hospital, University Health Network, and
University of Toronto, 3Department of Molecular Neurobiology, Max Planck Institute of
Experimental Medicine, Hermann-Rein-Str. 3D, 37075, Goettingen, Germany
*Correspondence:
Dr. Daniela Rotin
The Hospital for Sick Children
PGCRL 19-9715, 686 Bay St.
Toronto, Ont, Canada, M5G 0A4
Tel: (416) 813-5098
FAX: (416)813-5028
Email: [email protected]
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SUPPLMENTARY FIGURE LEGENDS
Supplementary Figure 1: Comparison of tumor numbers in Apc+/min tumors in the absence
or presence of Nedd4.
Comparison between the number of intestinal tumors formed in (a) Nedd4+/gt;Apc+/min mice and
Nedd4+/+;Apc+/min control littermates (p=0.0668, Wilcoxon&Mann-Whitney test), or in (b) VilCre;Nedd4fl/gt;Apc+/min
mice
and
Nedd4fl/+;Apc+/min
control
littermates
(p=0.0945,
Wilcoxon&Mann-Whitney test). Values are average number of tumors per mouse (meanSEM
of the indicated number of mice, in brackets). All mice represent crosses of Apc+/min mice to the
indicated Nedd4 mutants, as described in Fig 2.
Supplementary Figure 2: Lack of increased Apc+/min tumor number or size in the small
intestine upon loss/reduction of Nedd4 in the intestinal epithelium and surrounding tissue.
Tumor numbers (a) and size (b, surface area) from the small intestine of 15-17 weeks old VilCre;Nedd4fl/gt;Apc+/min mice compared to Nedd4fl/+;Apc+/min control littermates. Values are
average number of tumors per mouse or of tumor size (meanSEM) of the indicated number of
mice (in brackets). P= 0.20 for (a) and p=0.24 for (b). (Student’s t-test).
Supplementary Figure 3: Increased crypts length in Nedd4 KO tumors.
The length of 88 crypts (44 from each of the Control (Ctrl) and Nedd4 KO tumors) located in the
lumen side of the intestine was measured. The average ( SEM) of the crypt length is shown
(p<0.0001, Wilcoxon&Mann-Whitney test).
Supplementary Figure 4: Lymph nodes presence in tumor-bearing intestines.
Similar number of lymph nodes in the colons of the Nedd4fl/+;Apc+/min and VilCre;Nedd4fl/tg;Apc+/min mice. (a) Quantitation (MeanSEM) of the indicated number of lymph
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nodes from 10 FFPE intestinal sections from each of 10 mice per treatment. (p=0.7069,
Wilcoxon&Mann-Whitney test, N=10, n=10). (b) Examples of intestinal cross-sections used to
analyze lymph nodes number. Yellow arrows demarcate lymph nodes.
Supplementary Figure 5: A reduction in Goblet cells numbers in Nedd4 KO tumors
Nuclei in each of the crypts are counted and the percentage of goblet cells per crypt calculated.
The average % of Goblet cells from KO crypts (n=60) and control (Ctrl) crypts (n=109) are
shown. (Average  SEM, n= 60 crypts from Nedd4 KO tumor and 109 crypts from the control
tumor). ****p<0.0001, Wilcoxon&Mann-Whitney test). KO: Vil-Cre;Nedd4fl/gt; Apc+/min. Ctrl:
Nedd4fl/+; Apc+/min . Bottom panels depict representative crypt areas.
Supplementary Figure 6: Expression levels of WNT signaling genes in Nedd4 KO and
control (Ctrl) intestinal tumors
Mouse WNT Signaling Pathway qPCR Array in 96-well format was used to detect transcription
levels of genes in the WNT signaling pathway. A total of 84 genes were tested from either Nedd4
KO and Ctrl intestinal tumors. Gene names and fold changes (≥ 1.5-fold) are circled. Thicker
circle indicates a  2-fold change. Genes with grey background are excluded due to failure in the
qPCR quality control test with an undetectable expression.
Supplementary Figure 7: FL-LEF1 and YY1 expression upon loss of two Nedd4 alleles
relative to one allele in the intestinal epithelium.
Colonic tumors from Nedd4 heterozygote littermates with either one (Nedd4fl/tg;Apc+/min) or both
(Vil-Cre;Nedd4fl/tg;Apc+/min) Nedd4 alleles deleted from the intestinal epithelium were analyzed
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for LEF1-FL and YY1 expression by immunoblotting. Data of littermates are shown. Three
litters (8 mice) were analyzed in total. GAPDH was used as a loading control.
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