Knockdown of OTX2 in Medulloblastoma Cell Lines

OTX2 knock down in HDMB03 and MB3W1 G3 MB cells (2×10⁵ cells/well) was performed as previously described^{33 (link),34 (link)}. Briefly, OTX2 was silenced using 30 nM Silencer Select siRNAs 9931 or 9932 (Life Technologies). A non-silencing (scramble) served as the negative control. For bulk RNA sequencing, OTX2 was knocked down in three independent biological replicates for each cell line and silencing was confirmed by western blot (OTX2, Abcam, ab21990, rabbit, at 1:500, and β-actin, Sigma-Aldrich, A2228, mouse, at 1/1000 was used as a loading control) 72 hours following transfection. Total RNA was extracted from all samples using the Norgen RNA extraction kit (Norgen Biotek), and bulk RNA sequencing (RNAseq) was performed by StemCore laboratories at the Ottawa Hospital Research Institute (Ottawa, ON, Canada). For single-nucleus RNA sequencing (snRNAseq), the above was repeated but using 4.5X10⁵cells/well for MB3W1 and only one replicate was performed. Granule neuron differentiation was validated by western blot for RBFOX3 (NeuN) (Cell Signaling Technology, D4G4O, at 1:1000).

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Johjima T., Itoh N., Kabuto M., Tokimura F., Nakagawa T., Wariishi H, & Tanaka H. (1999). Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium. Proceedings of the National Academy of Sciences of the United States of America, 96(5), 1989-1994.

Publication 2022

Silencer ab21990 β-actin Norgen RNA extraction kit

Actin Biological Bulk Cell line Cells Granule Knock Mouse Neuron Rabbit Replicate Single nucleus Sirnas Transfection Western blot

Corresponding Organization :

Other organizations : Hospital for Sick Children, SickKids Foundation, University of Manitoba, Centre National de la Recherche Scientifique, Université Paris-Saclay, Université Paris Sciences et Lettres, Inserm, Université Paris-Sud, Institut Curie, University of Toronto, Princess Margaret Cancer Centre, University Health Network, Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, McGill Genome Centre, McGill University, Heidelberg University, German Cancer Research Center, Seoul National University Children's Hospital, Children's Memorial Health Institute, Centre de Recherche en Neurosciences de Lyon, Université Claude Bernard Lyon 1, Erasmus MC, Masaryk University, Johns Hopkins Medicine, Johns Hopkins University, Mayo Clinic in Arizona, Fred Hutch Cancer Center, Semmelweis University, Neurological Surgery, University of California, San Francisco, Asan Medical Center, University of Ulsan, Hospital Sant Joan de Déu Barcelona, Chinese University of Hong Kong, University of Pittsburgh, Centro Medico Nacional Siglo XXI, University of Alabama at Birmingham, Virginia Commonwealth University, Osaka National Hospital, University of Colorado Denver, Vanderbilt University Medical Center, Washington University in St. Louis, Kitasato University, Chonnam National University Hwasun Hospital, McMaster University, University of Naples Federico II, Santobono Children's Hospital, Case Western Reserve University, Emory University, Fondazione IRCCS Istituto Nazionale dei Tumori, Universidade de São Paulo, Centro Hospitalar Lisboa Norte, Hospital de Santa Maria, St. Jude Children's Research Hospital, University of Calgary, Hôpital Necker-Enfants Malades, Assistance Publique – Hôpitaux de Paris, Carlo Forlanini Hospital, Duke University, University of Geneva, Istituto Giannina Gaslini, Hospital Infantil de México Federico Gómez, Instituto Nacional de Pediatria, UPMC Hillman Cancer Center, Ontario Institute for Cancer Research, University of British Columbia, Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute, McGill University Health Centre, Cedars-Sinai Medical Center

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Variable analysis

independent variables

OTX2 knockdown using Silencer Select siRNAs 9931 or 9932 (Life Technologies)

dependent variables

Gene expression changes measured by bulk RNA sequencing (RNAseq)
Granule neuron differentiation measured by Western blot for RBFOX3 (NeuN)

control variables

Cell lines: HDMB03 and MB3W1 G3 MB cells
Cell density: 2x10^5 cells/well for bulk RNAseq, 4.5x10^5 cells/well for single-nucleus RNAseq
Incubation time: 72 hours following transfection

controls

Negative control: Non-silencing (scramble) siRNA

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