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,*

1Cell 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:

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)Vil-Cre;Nedd4fl/gt;Apc+/min mice andNedd4fl/+;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 oldVil-Cre;Nedd4fl/gt;Apc+/minmice 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 colonsof theNedd4fl/+;Apc+/min and Vil-Cre;Nedd4fl/tg;Apc+/min mice. (a) Quantitation (MeanSEM) of the indicated number of lymph 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 Figure6: 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 Figure7: 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 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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