Fig. 2
OPA1 requires ATP synthase activity to prevent mitochondrial ΔpH loss caused by CIII inhibition. a Representative color-coded frames from real time imaging of mtSypHer ratio fluorescence in MAFs of the indicated genotype. Where indicated, cells were incubated with oligomycin (oligo, 1 µM for 30 min) and antimycin A (AA) (10 µM added 5 min after oligo, for 25 min). Rainbow color bar: pseudocolor scale of SypHer fluorescence ratio. Scale bar, 20 µm. b Quantitative analysis of mitochondrial mtSypHer fluorescence ratio in real time imaging experiments as in a. Data are mean ± SEM of at least four independent experiments. c Representative pseudocolor-coded frames from real time imaging of TMRM fluorescence in MAFs of the indicated genotype. Where indicated, cells were incubated with oligomycin (1 µM for 5 min, before AA additions) and AA (2 µM for 25 min). Scale bar, 20 µm. d Quantitative analysis of mitochondrial TMRM fluorescence ratio in real time imaging experiments as in c. Where indicated, cells were treated with 2 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). Data are mean ± SEM of at least four independent experiments. e Representative color-coded images of mitochondrial mtSypHer fluorescence in Opa1flx/flx cells transfected with empty (EV) or Cre-encoding vectors and treated for 30 min with AA (10 µM) and oligomycin (1 µM, added 5 min before AA) where indicated. Rainbow color bar: pseudocolor scale of mtSypHer fluorescence ratio. Scale bar, 20 µm. f Quantitative analysis of mitochondrial mtSypHer fluorescence ratio in real time imaging experiments as in e. Data are mean ± SEM of at least 4 independent experiments. g Pseudocolor-coded frames of mtATeam FRET fluorescence ratio in cells of the indicated genotype. Where indicated, cells were treated for 10 min with AA (10 µM). Rainbow color bar: pseudocolor scale of ATeam fluorescence ratio. Scale bar, 20 µm. h Quantitative analysis of mitochondrial mtATeam fluorescence ratio in real time imaging experiments as in g. Data are mean ± SEM of at least three independent experiments. i Mean ± SEM of mtATeam fluorescence ratio slopes, calculated from experiments as in g. Where indicated, oligomycin (1 µM) was added 5 min before AA. *p < 0.05 in an unpaired two-sample Student’s t test
