REEP5 and MFN1/2 interaction regulates mitochondrial ROS level. The fluorescence ratio of 405 and 488 nm excitation was shown by heatmap. (A) Time course of roGFP2 fluorescence excitation (405/488 nm) ratiometric images (R405/488) in mitochondria of mito-roGFP2 stable U2OS cells after the addition of 200 µM H2O2, 1 mM DTT, or H2O2 + DTT for the indicated time. The ratiometric from indicated time points were calculated and shown in the graph. Bar: 10 µm. (B) Schematic workflow showing the internal control cells (labeled by #) were respectively mixed with either Ctrl siRNA (labeled by *) or REEP5 siRNA (labeled by *) transfected cells. The corresponding ratiometric images of mito-roGFP2 are shown below. Bar: 20 µm. (C) Quantification of mitochondrial ROS level from B. (D) Time course of HyPer7 fluorescence excitation (488/405 nm) ratiometric images (R488/405) of mito-HyPer7 stable U2OS cells exposed to 4 µM H2O2, 20 mM DTT, or H2O2 + DTT for the indicated time. Bar: 10 µm. The ratiometric changes overtime were calculated and shown in the graph. (E) Same as B, images of HyPer7 fluorescence excitation (488/405 nm) ratio. Bars: 20 µm. (F) Quantification of mitochondrial ROS level from E. (G) Wild-type and MFN1&2 DKO MEFs stably expressing mito-HyPer7 were mixed as in the schematic workflow, images of mitochondrial ROS are shown below. Bar: 20 µm. (H) Quantification of mitochondrial ROS level from G. (I) Schematic workflow and images of mitochondrial ROS changes upon rapamycin-induced REEP5-MFN2 binding. 40 nM Rapamycin was added at 0 min. Bars: 20 µm (whole cell view); 10 µm (zoomed-in images). (J) Quantification of mitochondrial ROS level from I. Values shown are means ± SEM, n = 6 for A; n = 7 for D; n = 25 for C; n = 22 for F; n = 24 for H; n = 7 for J.
