Partial ETC-supported mitoflash biogenesis in state II/IV respiration. (A) Experimental design. Complex IV substrates (2.5 mM ascorbate and 0.5 mM TMPD) were present without ADP. The black arrow shows the forward electron transfer pathway, and the dashed arrow shows the RET pathway. Different ETC inhibitors were also added as designated in different experimental groups (the concentrations of inhibitors were the same as in Fig. 1, and piericidin A [PieA, 1 µM] was also used for Complex I and myxothiazol [Myx, 1 µM] for Complex III). (B) Averaged trace of mitoflashes reported by cpYFP (n = 45 events). (C) Statistics of mitoflash frequency. Data are mean ± SEM, n = 14–134 image series from three to five mice for each group. **, P < 0.01; ***, P < 0.001 versus control group. (D) 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 files from three mice for each trace.