SiRNA SOD2 knockdown restores the sensitivity TrkAIII SH-SY5Y to agents that induce mitochondrial ROS production.

<p>A) Western blot demonstrating knockdown of SOD2 but not α-tubulin protein expression in TrkAIII SH-SY5Y cells transiently transfected for 48 hours with SOD2-specific but not with control siRNA, prior to assay. B) Digital fluorescence micrographs demonstrating Mitosox-reactive ROS (red) in TrkAIII SH-SY5Y cells cultured in the absence of siRNAs or ROS-inducing reagents (Control); treated with 25 nM control SiRNA or 25 nM SOD2 siRNA alone; or treated with either 25 nM control SiRNA or 25 nM SOD2 siRNA followed by 12 hours with 1 µM Rotenone or 250 µM Paraquat, or 3 hours with 1 µM LY83583 (bar = 100 µm). C) Histogram demonstrating differences in ROS levels in TrkAIII SH-SY5Y cells treated with control 25 nM siRNA-treated (Con SiRNA) or 25 nM SOD2 siRNA, alone; or for 12 hours with 1 µM Rotenone (Rot), 250 µM Paraquat or for 3 hours with 1 µM LY83583 (LY835), following 48 hours treatment with either control siRNA or SOD2 siRNA. Results are expressed as mean±s.d densitometric units per 100 cells, in three independent experiments performed in duplicate. Mean values are provided above each column and asterisks denote statistical significance by Student’s t-test (p<0.0005, df = 10) and One-Way ANOVA ((2, 15) p<0.0001), comparing inhibitor alone, ROS-inducer alone and inhibitor plus ROS-inducer groups.