The Netherlands Cancer Institute

4 ARTICLES PUBLISHED IN JoVE

Biology

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

David Svilar^1,2, Conchita Vens³, Robert W. Sobol^1,2,4

¹Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, ²Hillman Cancer Center, University of Pittsburgh Cancer Institute, ³Department of Experimental Therapy, The Netherlands Cancer Institute, ⁴Department of Human Genetics, University of Pittsburgh School of Public Health

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Biochemistry

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Misbha Ud Din Ahmad¹, Alexander Fish¹, Jeroen Molenaar¹, Sridhar Sreeramulu², Christian Richter², Nadide Altincekic², Harald Schwalbe², Hans Wienk¹, Anastassis Perrakis¹

¹Oncode Institute and Division of Biochemistry, the Netherlands Cancer Institute, ²Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Biology

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Dimitrios Laskaris^*1,2, Maria Azkanaz^*1,2, Mijke A. de Vreij-Kruidenier³, Doreen van Rijswoud-Ram³, Hendrik A. Messal^1,2, Jacco van Rheenen^1,2

¹Department of Molecular Pathology, The Netherlands Cancer Institute, ²Oncode Institute, ³Animal Laboratory Facility, The Netherlands Cancer Institute

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

Developmental Biology

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

Naomi J. M. Kakiailatu¹, Laura E. Kuil^1,2, Eric Bindels³, Joke T. M. Zink³, Michael Vermeulen³, Veerle Melotte⁴, Maria M. Alves¹

¹Department of Clinical Genetics, Erasmus University Medical Center - Sophia Children's Hospital, ²Division of Psychosocial Research and Epidemiology, the Netherlands Cancer Institute, ³Department of Hematology, Erasmus Medical Center, ⁴Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.