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Université de Lyon

11 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon
Benoît Drogue 1, Philippe Thomas 2, Laurent Balvay 3, Claire Prigent-Combaret 1, Corinne Dorel 4
1UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Université de Lyon, 2Département Biosciences, INSA de Lyon, Université de Lyon, 3INSERM U758, Ecole Normale Supérieure de Lyon, Université de Lyon, 4Laboratoire de Génie Civil et Ingénierie Environnementale, INSA de Lyon, Université de Lyon

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

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Biology

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells
Johann Gout 1,2,3,4,5, Roxane M. Pommier 1,2,3,4,5, David F. Vincent 1,2,3,4,5, Bastien Kaniewski 1,2,3,4,5, Sylvie Martel 1,2,3,4,5, Ulrich Valcourt *1,2,3,4,5, Laurent Bartholin *1,2,3,4,5
1INSERM U1052, Centre de Recherche en Cancérologie de Lyon, 2CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, 3Université de Lyon, 4Université Lyon 1, 5Centre Léon Bérard

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

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Medicine

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Philippe Pérot 1,2, Valérie Cheynet 1,2, Myriam Decaussin-Petrucci 1,3,4, Guy Oriol 1,2, Nathalie Mugnier 5, Claire Rodriguez-Lafrasse 1,4,6, Alain Ruffion 1,4,7, François Mallet 1,2
1Joint Unit Hospices de Lyon-bioMérieux, 2Medical Diagnostic Discovery Department, BioMérieux, 3Department of Pathology and Cytology, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, 4Medical Faculty, Lyon 1 University, 5Data and Knowledge Laboratory, BioMérieux, 6Department of Biochemistry and Molecular Biology, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, 7Department of Urology, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

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Medicine

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics
Marion Gilormini 1, Anne-Sophie Wozny 1,2, Priscillia Battiston-Montagne 1, Dominique Ardail 1,2, Gersende Alphonse 1,2, Claire Rodriguez-Lafrasse 1,2
1UCBL1, UMR/CNRS 5822, Laboratoire de Radiobiologie Cellulaire Moléculaire, Université de Lyon, 2Hospices-Civils-de-Lyon, Centre Hospitalier Lyon-Sud

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

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Cancer Research

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay
Jean-Baptiste Guy *1,2, Sophie Espenel *1,2, Alexis Vallard 1,2, Priscillia Battiston-Montagne 1, Anne-Sophie Wozny 1,3, Dominique Ardail 1,3, Gersende Alphonse 1,3, Chloé Rancoule 1,2, Claire Rodriguez-Lafrasse 1,3, Nicolas Magne 1,2
1UMR CNRS 5822 /IN2P3, IPNL, PRISME, Laboratoire de Radiobiologie Cellulaire et Moléculaire, Faculté de Médecine Lyon-Sud, Université Lyon 1, 2Département de Radiothérapie, Institut de Cancérologie de la Loire - Lucien Neuwirth, 3Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

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Chemistry

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis
Lukasz Bozycki 1, Magdalena Komiazyk 1, Saida Mebarek 2,3,4,5,6, Rene Buchet 2,3,4,5,6, Slawomir Pikula 1, Agnieszka Strzelecka-Kiliszek 1
1Laboratory of Lipid Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 2Université de Lyon, 3Université Lyon 1, 4L'insitut National des Sciences Appliquées (INSA) de Lyon, 5Ecole Supérieure de Chimie Physique Electronique (CPE) Lyon, 6Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR), L'institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS)

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

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Developmental Biology

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues
Simone Bovio 1,2, Yuchen Long 2, Françoise Monéger 2
1SFR Biosciences, Université de Lyon, 2Laboratoire de Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCBL, INRA, CNRS

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

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Cancer Research

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence
Jessica Garcia 1,2,3, David Barthelemy 1,2,3, Florence Geiguer 1,2, Julie Ballandier 1,2,3, Kathryn W. Li 4, Jean-Phillippe Aurel 4, Frédérique Le Breton 5, Claire Rodriguez-Lafrasse 1, Brigitte Manship 3, Sébastien Couraud 6,7, Léa Payen 1,2,3
1Laboratoire de Biochimie et Biologie Moléculaire, Groupe Hospitalier Sud, Hospices Civils de Lyon, 2Circulating Cancer (CIRCAN) Program, Hospices Civils de Lyon Cancer Institute, 3University of Lyon, Claude Bernard University, Cancer Research Center of Lyon, 4Biolidics Limited, 5Institut de Pathologie Multisites des HCL-Site Sud, Hospices Civils de Lyon, 6Acute Respiratory Disease and Thoracic Oncology Department, Lyon Sud Hospital, Hospices Civils de Lyon Cancer Institute, 7EMR-3738 Therapeutic Targeting in Oncology, Lyon Sud Medical Faculty, Lyon 1 University

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

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Biology

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models
Alejandro Lopez Gonzalez *1, Léa Luciana *1, Clémentine Le Nevé 2, Julie Valantin 2, Laura Francols 2, Nicolas Gadot 2, Christophe Vanbelle 3, Laurianne Davignon 4, Laura Broutier 1
1Childhood Cancer & Cell Death (C3), Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), Lyon 69373, 2Plateforme Anatomopathologie Recherche, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), 3Plateforme d’Imagerie cellulaire, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon (CRCL), 4PerkinElmer S.A.S.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 µm to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

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Cancer Research

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue
Coralie Poulard *1,2,3, Julien Jacquemetton *1,2,3, Julie Valantin 1,2,3,4,5, Isabelle Treilleux 1,2,3,4, Muriel Le Romancer 1,2,3
1Université de Lyon, 2Inserm U1052, Centre de Recherche en Cancérologie de Lyon, 3CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, 4Pathology Department, Centre Leon Bérard, 5Plateforme Anatomopathologie Recherche

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

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Biology

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts
Kawtar Arizkane 1,2,3, Kevin Geistlich 1,2,3,4, Laurine Moindrot 1,2,3, Emma Risson 1,2,3, Sandrine Jeanpierre 1,2,3,4, Léa Barral 1,2,3, Anaëlle Bobard 1,2,3, Giulio Menegazzi 5, Thibault Voeltzel 1,2,3, Véronique Maguer-Satta 1,2,3, Sylvain Lefort 1,2,3
1Centre de Recherche en Cancérologie de Lyon, 2Centre de Recherche en Cancérologie de Lyon, 3Université de Lyon, 4Centre Léon Bérard, 5Department of Experimental and Clinical Biomedical Sciences Mario Serio, Università degli Studi di Firenze

Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events.

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