Figure 5

Pharmacological inhibitors of NADPH oxidase and silencing p22 ^phox using siRNA attenuate MPP+ induced ROS. N27 cells were treated for 18 hours with 300 μM MPP+ and increasing concentrations of either apocynin (A) or phenylarsine oxide (PAO) (B). H₂O₂ levels were measured using caboxy-H₂-DCFDA and flow cytometry. ROS levels are represented as percent of MPP+ induced ROS. * represents p < 0.05 compared to cells receiving no inhibitor. (C) N27 cells were transfected with a non-targeting control (NTC) siRNA or a SmartPool siRNA targeting p22^phox. Total RNA was collected and reverse transcribed to cDNA. Primers complimentary to rat p22^phox were used to amplify the cDNA. An image of a single representative ethidium bromide-stained agarose gel is shown from one knockdown experiment out of three that produced an average knockdown of 40%. (D) N27 cells were transfected with NTC or p22^phox siRNA Smartpool and the intracellular H₂O₂ was measured with flow cytometry as described above. * represents p < 0.05 compared to NTC siRNA-treated cells. Data are from 3 independent experiments with n = 6 wells per experiment.