PCNA and DNA pol-β are required for neuronal death induced by MPP⁺.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) Apoptotic rate determined by Hoechst staining. (<b>D</b>) Representative images of cells after Hoechst staining. Arrows indicate the apoptotic cells. (<b>E</b>) 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. (<b>F</b>) 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. (<b>G</b>) 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.