Fig. 1.

Analysis of levels of mtDNA mutations in tissues and characterization of ROS production and respiratory chain function in MEFs. (A) The levels of mtDNA mutations (mut; cytochrome b region) in wild-type (wt; blue bars) and mtDNA mutator (red bars) embryo heads and mouse brains at different time points. Bars show mean values, and error bars indicate the SD. The low background level of PCR-induced mutations has been subtracted. (B) FACS analysis of production after 30 min (thin lines) and 120 min (thick lines) incubation with dihydroethidium in primary cultures of MEFs from wild-type (blue lines) and mtDNA mutator (red lines) embryos. (C) FACS analysis of H₂O₂ production after 30 min incubation with carboxy-H₂DCFDA in wild-type (blue line) and mtDNA mutator (red line) primary MEF cultures. (D) FACS analyses of production after 30 min incubation with dihydroethidium in wild-type (blue line) and mtDNA mutator (red line) immortalized MEFs and primary MEFs from mtDNA mutator embryos (yellow line). The black line in all FACS analyses indicate negative control, i.e., wild-type cells analyzed without previous treatment with dihydroethidium or H₂DCFDA. FL1-H and FL2-H, fluorescence intensity in log scale (green and red fluorescence, respectively). (E) Polarographic investigation of respiratory chain function in immortalized MEFs from wild-type (blue bars) and mtDNA mutator (red bars) embryos. Measurements (mean values ± SD) were performed without addition of exogenous substrate (rotenone-sensitive respiration) and with addition of succinate, with or without uncoupler. Asterisks indicate level of statistical significance: *, P < 0.05; **, P < 0.01.