Full ETC-supported mitoflash biogenesis in state II/IV respiration. (A) Top: Experimental design. Complex I substrates (5 mM glutamate and 5 mM malate) without ADP were added to allow state II/IV respiration. The black arrow shows the electron transfer pathway. IMS, intermembrane space. ETC inhibitors targeting Complex I (5 µM rotenone [Rot]), Complex II (5 mM malonate [Mal]), or 50 nM atpenin A5 [A5]), Complex III (5 µg/ml antimycin A [AA]), or Complex IV (5 mM KCN or 5 mM NaN₃) were used as designated. Bottom: Average of mitoflash traces reported with cpYFP (n = 33 events). (B) Effect of ETC inhibitors on mitoflash frequency supported by Complex I substrates. Data are mean ± SEM, n = 13–94 image series from three mice for each group. ***, P < 0.001 versus control group. (C) Averaged time courses of ΔΨ_m measured with TMRM in response to addition of ETC inhibitors. Arrow indicates the time of inhibitor administration. n = 3 image series from three mice for each group. Error bars are omitted for clarity. (D–F) As in A–C except that a Complex II substrate (2.5 mM succinate) was used to replace Complex I substrates. n = 61 events for the averaged mitoflash trace in D. Data are mean ± SEM, n = 13–39 image series from three to eight mice for each group in E, ***, P < 0.001 versus control group. n = 3 image files from three mice for each trace in F. Cyt c, cytochrome c.