Supplementaryfigure Legends and Captions for CAN-14-1726R1 Diefenbacher Et Al

SupplementaryFigure legends and captions for CAN-14-1726R1 Diefenbacher et al.

Supplementary Fig. S1. Loss of Usp28 ameliorates the Fbw7 inactivation phenotype and partly restores cell differentiation in the intestine.

(A) qRT-PCR analysis of Usp28 and Fbw7 mRNA from isolated crypt cells of wild type C57BL/6 (WT), Usp28∆IEC, Fbw7∆IEC and Fbw7∆IEC; Usp28∆IEC animals. Data represent means; n = 5 animals pooled per group.

(B) qRT-PCR analysis of progenitor and differentiation marker gene expression in WT, Fbw7∆IEC and Fbw7∆IEC; Usp28∆IEC animals. Data represent means ± SEM; n = 5/group; *p < 0.05; n.s., not significant.

Supplementary Fig. S2. Deletion of Usp28 prolongs life expectancy and reduces tumor burden in the Fbw7-deleted APCmin/+intestinal tumour model.

(A) Average survival of mice with the indicated genotypes. APCmin/+; Fbw7∆IEC; Usp28∆IEC animals survive significantly longer than APCmin/+; Fbw7∆IEC animals (*p=0.0188). Error bars indicate SEM.

(B) Gut roll sections of small bowel (Duodenum, Jejunum, Ileum) and colon stained with H&E, showing tumors within the intestine of APCmin/+; Fbw7∆IEC and APCmin/+; Fbw7∆IEC; Usp28∆IEC animals. Images are representative of at least 5 animals per genotype. Dashed lines highlight tumors. Scale bar represents 5mm. Age of animals is indicated.

Supplementary Fig. S3. The interaction between Usp28 and its substrate c-Myc is independent of Fbw7.

Co-immunoprecipitation of endogenous c-Myc with Usp28 in KRasG12D; p53∆/∆(KP) and KP Fbw7∆/∆ cell lines.

Supplementary Fig. S4. Schematic representationof Usp28-Fbw7-substrate crosstalk in Fbw7-proficient and deficient cells and speculative model of the consequencesof this crosstalk in the intestinal crypt.

(A) Schematic showing the possible modes of interaction of Usp28 with its substrate in the presence and absence of Fbw7.

(B) (Left) Histology showing opposing effects of Fbw7 and Usp28 on the extent of c-Jun expression in the murine intestinal crypt. (Right) Speculative model of how Fbw7 and Usp28 may achieve the correct balance of proliferation and differentiation in the crypt, based on opposing regulation of their shared substrates.

- 1 -