Collablots: Quantification of Collagen VI Levels and Its Structural Disorganisation in Cell Cultures From Patients With Collagen VI-Related Dystrophies
Por:
Osegui-Barcenilla N, Sendino M, Martín-González S, González-Moro I, Benito-Agustino A, Torres-Conde N, López-Martínez A, Jimenez-Mallebrera C, López-Marquez A and Arechavala-Gomeza V
Publicada:
22 may 2025
Resumen:
AimsThis study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies.MethodsWe developed a combined in-cell western (ICW) and on-cell western (OCW) assay, which we have called 'collablot', to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen-hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures.ResultsCollablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls.ConclusionsThe collablot assay represents a suitable method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.
Filiaciones:
Osegui-Barcenilla N:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
Sendino M:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
Martín-González S:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
González-Moro I:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
Benito-Agustino A:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
Torres-Conde N:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
López-Martínez A:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
Jimenez-Mallebrera C:
Laboratory of Applied Research in Neuromuscular Diseases, Neuromuscular Pathology Unit, Neuropediatric Service, Institut de Recerca Sant Joan de Déu, Esplugues de Llobregat, Spain
Rare Diseases Network Biomedical Research Center (CIBERER), Madrid, Spain
López-Marquez A:
Laboratory of Applied Research in Neuromuscular Diseases, Neuromuscular Pathology Unit, Neuropediatric Service, Institut de Recerca Sant Joan de Déu, Esplugues de Llobregat, Spain
Rare Diseases Network Biomedical Research Center (CIBERER), Madrid, Spain
Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Barcelona, Spain
Arechavala-Gomeza V:
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain
Ikerbasque, Basque Foundation for Science, Bilbao, Spain
Biobizkaia Hlth Res Inst, Nucl Acid Therapeut Rare Disorders NAT RD,
Baracaldo, Spain
Inst Recerca St Joan de Deu, Neuropediat Serv, Neuromuscular Pathol
Unit, Lab Appl Res Neuromuscular Dis, Esplugas de Llobregat, Spain
Rare Dis Network Biomed Res Ctr CIBERER, Madrid, Spain
Univ Barcelona, Fac Biol, Dept Genet Microbiol & Stat, Barcelona, Spain
Basque Fdn Sci, Ikerbasque, Bilbao, Spain
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