Fig. 2: Examination of iron-induced ROS production.

Using CellROX Deep Red to detect ROS we performed confocal microscopy analysis with time-lapse acquisition at 30 min intervals up to 4 h A in the presence or absence of iron (250 µM FeCl₃) with quantitation shown in B (n = 3). The effect of anti-oxidant 100 µM MnTBAP for 30 min prior to iron treatment on iron-induced ROS production was assessed using CellROX Deep Red after treatment of cells with 250 µM FeCl₃ for 4 h by confocal microscopy C (n = 3). D Representative plots from one experiment showing frequency of cell populations in oxidized (mROShi GSHlo), intermediate (mROSint GSHhi) and reduced (mROSlo GSHhi) states based on intracellular levels of glutathione (GSH; measured by mBBr) and mitochondrial ROS levels (mROS; measured by MitoSox Red) upon treating cells with 250 µM FeCl₃ for 4 h (Iron) in the presence or absence of 100 µM MnTBAP or 20 nM SKQ1. Graphs show percentages of cells in highly oxidized E, reduced F, and intermediately oxidized G states from three experiments as in D. H Graphs show the average mROS fluorescence per cell from three experiments as in D (n = 3). I Representative plots showing frequency of cell populations with depolarized (JC-1 monomers) and polarized mitochondria (JC-1 aggregates) upon treating with 250 µM FeCl₃ for 4 h (Iron) in the presence or absence of 100 µM MnTBAP or 20 nM SKQ1. Graphs show percentages of cell populations with depolarized J and polarized K from three experiments as in I (n = 3). Scale bar denotes 50 µm. All graphs show mean ± SEM and * = P < 0.05, ** = P < 0.01, *** = P < 0.001 versus control, # = P < 0.05, ## = P < 0.01, ### = P < 0.001 versus iron.