European Institute of Oncology View Institution's Website 7 articles published in JoVE Biochemistry An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA Núria Crua Asensio1, Stefano Rizzieri2, Alessandro Cuomo2, Gian Gaetano Tartaglia1,3, Elsa Zacco1 1Center for Human Technology, Italian Institute of Technology (IIT), 2Department of Experimental Oncology, European Institute of Oncology (IEO), 3Department of Biology and Biotechnologies ‘Charles Darwin’, Sapienza University of Rome Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis. Biochemistry A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis Marianna Maniaci*1,2, Federica Marini*1, Enrico Massignani1,2, Tiziana Bonaldi1 1Department of Experimental Oncology, European Institute of Oncology IRCCS (IEO), 2European School of Molecular Medicine (SEMM), c/o Campus IFOM-IEO Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here. Medicine A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy (PRRT): 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods Maria Luisa Belli1, Emilio Mezzenga1, Valentina Di Iorio2, Monica Celli3, Paola Caroli3, Elisabeth Canali3, Federica Matteucci3, Elisa Tardelli3, Ilaria Grassi3, Maddalena Sansovini3, Silvia Nicolini3, Stefano Severi3, Marta Cremonesi4, Mahila Ferrari5, Giovanni Paganelli3, Anna Sarnelli1 1Medical Physics Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy, 2Oncology Pharmacy, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy, 3Nuclear Medicine Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy, 4Radiation Research Unit, Istituto Europeo di Oncologia (IEO) IRCCS, Milano, Italy, 5Medical Physics Unit, Istituto Europeo di Oncologia (IEO) IRCCS, Milano, Italy This method estimates the absorbed dose of different structures for peptide-receptor-radionuclide-therapy (PRRT) with the possibility of avoiding organ overlap on 2D-projections. Serial whole-body planar images permit estimation of mean absorbed doses along the whole body, while the hybrid approach, combining planar images and 3D-SPECT/CT image, overcomes the limitations of structure overlapping. Biology Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis Moreno Zamai1, Antonio Trullo2, Elvira Arza1, Ugo Cavallaro3, Valeria R. Caiolfa1,2 1Unit of Microscopy and Dynamic Imaging, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, 2Centro di Imaging Sperimentale, Ospedale San Raffaele, Milan, Italy, 3Unit of Gynecological Oncology Research, European Institute of Oncology IRCCS, Milan, Italy We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. Immunology and Infection Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses Fulvia Milena Cribiù*1, Claudia Burrello*2, Roberta Tacchi1, Francesca Boggio1, Dario Ricca1, Flavio Caprioli3,4, Stefano Ferrero1, Federica Facciotti2 1Pathology Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, 2Department of Experimental Oncology, European Institute of Oncology, 3 Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding human samples. Biology The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin Monica Soldi1, Tiziana Bonaldi1 1Department of Experimental Oncology, European Institute of Oncology By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains. Biology A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants Katerina Tsilingiri1, Angelica Sonzogni2, Flavio Caprioli3, Maria Rescigno1 1Department of Experimental Oncology, European Institute of Oncology, 2Department of Pathology and Laboratory Medicine, European Institute of Oncology, 3U.O. Gastroenterologia 2, IRCCS Ca' Granda, Ospedale Policlinico di Milano We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.