In vivo disruption of an Rb-E2f-ezh2 signaling loop causes bladder cancer Mirentxu Santos1, 2

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Supplementary Information of

In vivo disruption of an Rb-E2F-Ezh2 signaling loop causes bladder cancer

Mirentxu Santos1, 2*, Mónica Martínez-Fernández1, 2*, Marta Dueñas1, 2, Ramón García-Escudero1, 2, Begoña Alfaya1, Felipe Villacampa3, Cristina Saiz-Ladera1, Clotilde Costa1, Marta Oteo2, 4, José Duarte3, Victor Martínez3, Mª José Gómez-Rodriguez3, Mª Luisa Martín3, Manoli Fernández3, Patrick Viatour5,6, Miguel A Morcillo2, 4, Julien Sage5, Daniel Castellano3 Jose L Rodriguez-Peralto7, Federico de la Rosa3, and Jesús M Paramio1,2


1 Unidad de Oncología Molecular. CIEMAT (ed70A). Av Complutense 40. 28040 Madrid SPAIN

2 Unidad de Oncogenómica. Instituto de Investigación 12 de Octubre i+12, UCM. Av Cordoba s/n. 28041 Madrid SPAIN

3 Unidad de Uro-Oncología. Hospital Universitario 12 de Octubre. Av Cordoba s/n. 28041 Madrid SPAIN

4 Unidad De aplicaciones de Radiositótopos CIEMAT (ed 12). Av Complutense 40. 28040 Madrid SPAIN

5 Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA

6 Present address: Center for Childhood Cancer Research, The Children’s Hospital of Philadelphia, Dept. of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. USA

7 Servicio de Anatomía Patológica. Hospital Universitario 12 de Octubre. Instituto de Investigación 12 de Octubre i+12, UCM. Av Cordoba s/n. 28041 Madrid SPAIN

* These authors contributed equally to this work and are co-first authors.
This file contains:

Supplementary Methods

Supplementary Figure S1-S6 (and corresponding legends)

Supplementary Tables S1, S7, S8, S9, S13 and S14 (other tables are included as separate excel files)

Supplementary References


In vivo Detection of tumors

Computerized tomography was used to monitor in vivo tumor presence. The animals were sedated by isofluorane inhalation anesthesia (2-3% isofluorane, Fluovac device, Harvard Apparatus); next, they were given a tail vein injection of 0.2 ml Iopamire 300 (Laboratorios Farmaceuticos Rovi, Spain). CT data were acquired 30 minutes after contrast injection on an Argus PET-CT scanner (Suinsa, Madrid, Spain) using 45 kV X-rays with 150 µA current. Images were acquired from 360 projections over 360º degree rotation and were reconstructed using a FeldKamp algorithm.

Whole transcriptome analysis

RNA was purified as commented above and analyzed by Bioanalyzer electropherogram. Samples showing RIN number above 8 (10 normal and 28 Ta and T1 stage human tumor samples in our series) were selected for microarray analysis. Biochip normalization was done at the transcript level with RMA (1, 2). Supervised analysis of differential gene expression was performed using Student’s T test (Adjusted Bonferroni p-val≤0.01 using 1000 random permutations) using the Multiexperiment Viewer 4.5 (MeV 4.5) software (3). Hierarchical clustering analysis was done using Pearson correlation and complete linkage method. Probesets showing statistically significant differences were uploaded into DAVID Functional Annotation web tool, which computes enrichment of Gene Ontology biological processes terms using EASE score (4, 5). Oncomine Gene Expression Signatures database (6) was used to search for overlapping of the obtained gene signatures using Fisher's exact test, and was considered statistically significant for Odds Ratio ≥ 2 and p≤ 0.05. Predictive determination of transcription factor binding to the identified deregulated genes was performed using the Chromatin immunoprecipitation Enrichment Analysis (ChEA) tool (7). Gene Set Enrichment Analysis (GSEA) (8) was performed as reported (9). Datasets have been deposited in GEO (GSE38264). Mouse to human comparison was performed essentially as described elsewhere (10).

Supp Fig 1 Identification of bladder tumors by Computerized tomography (CT)

A-D) Representative CT image examples showing bladder filling (using iopamire as a contrast agent) demosntrating the presence of a superficial outgrowths (denoted by red arrow) from the bladder wall in a RbF/F;p130F/F;p107-/- mouse at different times after adenoCre inoculation: A=16 weeks, B=20 weeks, C=22 weeks D=26 weeks..

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