Mitochondrial dysfunction in cells with reduced mtDNA content. (a) Relative mtDNA content analyzed by real-time PCR amplification of mtDNA-coded COX I and nuclear-coded COX IV after EtBr treatment in MCF10A (left) and MCF 7 (right). (b, c) Cellular respiration indicated as the OCR of parental, mtDNA-reduced and reverted MCF10A (b) and MCF7 (c) cells measured by Seahorse XF24 analyzer using 50 000 cells in each type. Coupled and maximal respiration were determined by sequential addition of oligomycin (2 μg/ml), 3,5 DNP (75 μM) and rotenone (1 μM), respectively, as indicated in the figure. (d, e) Mitochondrial staining of MCF10A (d) and MCF7 (e) cells stained with cytochrome oxidase (CcoIVi1) antibody, and nuclei were stained with DAPI. Scale bar, 20 μm.