Method for depletion of mitochondria DNA in human bronchial epithelial cells.

Autor:	Novotny MV; Lerner Research Institute, USA., Xu W; Lerner Research Institute, USA., Mulya A; Lerner Research Institute, USA., Janocha AJ; Lerner Research Institute, USA., Erzurum SC; Lerner Research Institute, USA.; Respiratory Institute: Cleveland Clinic, 9500 Euclid Avenue, NB2-21, Cleveland, OH 44195, USA.
Jazyk:	angličtina
Zdroj:	MethodsX [MethodsX] 2023 Nov 24; Vol. 12, pp. 102497. Date of Electronic Publication: 2023 Nov 24 (Print Publication: 2024).
DOI:	10.1016/j.mex.2023.102497
Abstrakt:	Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to ∼95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 µM 2',3'-dideoxycytidin (ddC). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA . The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ∼two-fold in cells when mtDNA was eliminated. BET-1A ρ0 and BEAS-2B ρ0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mitochondrial DNA-depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis.•BET-1A and BEAS-2B cells were treated with ethidium bromide (EtBr) with or without 2',3'-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA).•Cells' mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR).•Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer. Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (© 2023 The Authors. Published by Elsevier B.V.)
Databáze:	MEDLINE
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