TrkAIII attenuation of mitochondrial ROS production is reversed by TrkA inhibitors.

<p>A) Digital fluorescence micrographs demonstrating Mitosox-reactive ROS (red) in untreated pcDNA, TrkA and TrkAIII SH-SY5Y cell lines (Control) or following treatment for 12 hours, at 37°C with: 1 µM Rotenone; 250 µM Paraquat; or for 3 hours at 37°C with 1 µM LY83583 (bar = 100 µm). B) Histograms showing densitometric fold difference in Mitosox reactive ROS levels in pcDNA, TrkA and TrkAIII SH-SY5Y cells following 12 hours treatment with 1 µM Rotenone (Rot); for 12 hours with 250 µM Paraquat (Par); or for 3 hours with 1 µM LY83583, compared to untreated controls (Con), given the arbitrary value of 1±s.d. Results are displayed as mean±s.d fold difference compared to untreated controls in three independent experiments. Mean values are provided above each column and asterisks denote statistical significance by t-test comparison of treated cell lines to untreated controls. C) Digital fluorescence micrographs demonstrating Mitosox-reactive ROS (red) in untreated TrkAIII SH-SY5Y cells (Control); TrkAIII SH-SY5Y cells treated for 36 hours with 1 µM CEP-701, 1 µM K252a, 1 µM Gö6976 or 100 nM GW441756, alone; TrkAIII SH-SY5Y cells treated for 12 hours with either 1 µM Rotenone, 250 µM Paraquat or for 3 hours with 1 µM LY83583, alone; and TrkAIII SH-SY5Y cells pre-incubated for 24 hours with either 1 µM Cep-701, 1 µM K262a, 1 µM Gö6976 or 100 nM GW441756, then treated for 12 hours with either 1 µM Rotenone, 250 µM Paraquat or for 3 hours with 1 µM LY83583, in the continuous presence of each respective inhibitor (bar = 100 µm). D) Histogram demonstrating the differences in ROS levels detected in untreated TrkAIII SH-SY5Y cells (Con); TrkAIII SH-SY5Y cells treated with 1 µM CEP-701 (CEP), 1 µM K252a (K252), 1 µM Gö6976 (Gö69), 100 nM GW441756 (GW4), 1 µM Rotenone (Rot), 250 µM Paraquat (Par) or 1 µM LY83583 (LY83), alone; or with 1 µM Rotenone (Rot), 250 µM Paraquat (Par) or 1 µM LY83583 (LY83), following pre-incubation with, and in the continuous presence of, 1 µM CEP-701, 1 µM K252a, 1 µM Gö6976 or 100 nM GW441756, as described for 5C. 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<0.0004, df = 10) and One-Way ANOVA ((2, 15) P<0.0001), comparing inhibitor alone, ROS-inducer alone and inhibitor plus ROS-inducer groups.