ATPase-supported mitoflash biogenesis. (A) Experimental design. ATP (3 mM) was present without any respiratory substrates. AMP-PNP and inhibitors were added as described to dissect the possible involvement of different ETC complexes and Complex V. (B) ATP but not AMP-PNP restored ΔΨ_m measured with TMRM. Traces were averaged from three image series. (C) Top: Averaged trace of concurrent cpYFP-reported mitoflashes in the presence of ATP (n = 52 events). Bottom: No mitoflashes were recorded in the presence of AMP-PNP (n = 22 mitochondria). (D) Effects of ETC and ATPase inhibitors on ATPase-supported mitoflash frequency. Note also that AMP-PNP alone failed to elicit any mitoflash activity. Data are mean ± SEM, n = 17–133 image series from three mice for each group. ***, P < 0.001 versus control group. (E) Response of ΔΨ_m measured with TMRM to ETC or ATPase inhibitors. Arrow indicates the time of inhibitor administration. n = 3 image series from three mice for each averaged trace. Error bars are omitted for clarity.