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. 2011 Jan 5;31(1):247–261. doi: 10.1523/JNEUROSCI.4589-10.2011

Figure 1.

Figure 1.

Cellular 2-Cys PRXs protect against 6-OHDA neurotoxicity in MN9D cells. A, Neuronal-differentiated MN9D cells were infected for 3 d with lentiviral vectors containing cDNA of human PRX1, PRX2, PRX3, PRX4, or the empty vector, and exposed to 6-OHDA at the indicated concentrations. Cell viability was determined at 24 h after 6-OHDA exposure. *p < 0.05, **p < 0.01 versus empty vector controls; n = 12 from 3 independent experiments. B, MN9D cells were transfected for 3 d with lentiviral vectors containing shRNA targeting PRX1 (PRX1t), PRX2 (PRX2t), PRX3 (PRX3t), PRX4 (PRX4t), or the PRX2 scramble control sequence (PRXsc), and exposed to 6-OHDA. Cell viability was determined at 24 h after 6-OHDA exposure. *p < 0.05, **p < 0.01 versus PRXsc controls; n = 12 from three independent experiments. C, Under the conditions of B, total cellular PRX activity was determined at 2 h after 6-OHDA (50 μm) or vehicle. *p < 0.05, **p < 0.01 versus PRXsc controls; data from four experiments. D, Nuclear staining (DAPI) and TUNEL staining at 24 h after 6-OHDA (50 μm) in MN9D cells infected with lentiviral vectors for PRX2 overexpression (PRX2) or knockdown (PRX2t). Note that PRX2 overexpression attenuates, whereas PRX2 knockdown promotes, nuclear apoptosis and DNA fragmentation (arrows) following 6-OHDA exposure. E, PRX2 overexpression attenuates, whereas PRX2 knockdown promotes, apoptotic DNA fragmentation 24 h after 6-OHDA exposure, as determined using DNA gel electrophoresis. The gel is representative of three experiments with similar results. F, PRX2 overexpression attenuates cytochrome c release at 4 and 16 h after 6-OHDA (50 μm) exposure, determined by subcellular fractionation and immunoblotting. Right, The graph illustrates the cytosolic cytochrome c after 6-OHDA in cells infected with Lenti-PRX2 or the control vector Lenti-GFP. *p < 0.05 versus noninfected controls; n = 4/condition. G, H, PRX2 overexpression attenuates the activation of caspase-9 and caspase-3 at 6 and 16 h after 6-OHDA (50 μm) exposure, determined by immunoblotting against cleaved caspases (G) and peptide substrate-based protease activity assays (LEHD for caspase-9, DEVD for caspase-3) (H), respectively. *p < 0.05; **p < 0.01 versus Lenti-GFP-infected controls, n = 4/condition. Cyto, Cytochrome; COX, cyclo-oxygenase.