PINK1 Deficiency Figure 6. Free Radical Production in PINK1 knockdown neurons. A) and (B) demonstrate a higher basal production of ROS production in mitochondria of PINK1 kd neurons compared to controls. Image in Figure 6A confirms mitochondrial localisation of Mitosox probe in neurons. Inhibition of complex 1 with rotenone induced a significant increase of ROS production in control cells. Inhibition of complex 1 in PINK1 kd cells slightly increased ROS production which is still higher than rotenone induced ROS production in control mitochondria. The basal rate of Mitosox fluorescence in control cells was taken as 100%. C) Shows an increase in the rate of HEt fluorescence in response to 50 mM KCL in control neurons. D) PINK1 kd neurons have higher basal rates of cytosolic ROS production compared to control neurons. Stimulation using KCl results in an increase in ROS production which is more marked in control than PINK1 kd neurons. E) Histogram showing decreased reduced and total glutathione concentrations in young (dd5 i) and in aged (dd43 ii) and human PINK1 kd neurons compared to controls. Values represent means 6sem from triplicates of three independent samples. doi:10.1371/journal.pone.0002455.g006 PLoS ONE | www.plosone.org 12 June 2008 | Volume 3 | Issue 6 | e2455