PCNA and DNA pol-β are required for neuronal death induced by MPP+.
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(A) Cell viability assay. The neurons were exposed to 200 µM MPP+ for 6 h, 12 h, and 24 h, respectively. Cell viability was determined using the MTT method. Data are expressed as a percentage of the value in untreated control cells. (B) Apoptosis rata determined by TUNEL staining. After the neurons were exposed to 200 µM MPP+ for 6 h, 12 h, or 24 h, they were immunostained with neuronal marker MAP2. Neuronal apoptosis was detected by TUNEL staining. (C) Apoptotic rate determined by Hoechst staining. (D) Representative images of cells after Hoechst staining. Arrows indicate the apoptotic cells. (E) Immunoblotting analysis of the expression of PCNA, DNA pol-β, DNA pol-λ, DNA pol-η, and active caspase-3 after treatment with MPP+. The protein levels of PCNA, DNA pol-β and active caspase-3 increased significantly after the neurons were exposed to 200 µM MPP+ for 12 h or 24 h. (F) Immunoblotting experiments showing the efficiency of PCNA and DNA pol-β shRNA. The neurons were infected with PCNA and/or DNA pol-β shRNA lentivirus for 24 h, and then exposed to 200 µM MPP+ for 12 h. The expression of PCNA and DNA pol-β was knocked down by their specific shRNAs. (G) Effect of PCNA and DNA pol-β knockdown on neuronal apoptosis was detected by TUNEL staining. Both shRNAs attenuated neuronal apoptosis induced by MPP+. Date represent the mean ± SEM from four independent experiments. *p<0.05, **p<0.01 compared with control group.