ROS inhibition increased mitochondrial membrane potential.

(A) Simulated time-courses for mitochondrial membrane potential (ψm). Gradual ROS inhibition from 0% (black, control) to 90% predicted an increase in mitochondrial ψm due to perturbation of the new mitochondrial population. (B) The model prediction was confirmed by measuring mitochondrial membrane potential by live cell imaging and quantifying the fluorescence intensities (n = 3). Exogenous addition of SOD and catalase significantly increased the average ψm (Mann-Whitney test, * P<0.05) in vitro. In silico inhibition of ROS levels also partially reactivated mitochondrial ψm in a dose dependent manner, with between 15 and 30% levels giving equivalent restoration of ψm to the in vitro data. In vitro mitochondrial ψm was determined in MRC5 cells 15, 18 and 21 days post IR using live cell imaging of cells loaded with the mitochondrial ψm dependent dye TMRM and non-potential dependent mitotracker green. (C) Example images of data used in (B) for control cells (upper panel) and cells treated with SOD and catalase (100 U each) in the medium (lower panel) for 15 days post IR, stained with the mitochondrial ψm dependent dye TMRM, the non-potential dependent dye mitotracker green, and the nuclear counterstain Hoechst 33342. Scale bar is 10 µm.