Method for depletion of mitochondria DNA in human bronchial epithelial cells

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 lack-ing. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to similar to 95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 mu M 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 similar to two-fold in cells when mtDNA was eliminated. BET-1A p0 and BEAS-2B p0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mito-chondrial DNA-depleted BET-1A p0 and BEAS-2B p0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis. center dot 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). center dot Cells' mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR). center dot Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer.