Research Paper Volume 12, Issue 24 pp 25035—25059

Figure 4. Knockdown of DOR or PINK1 interfered with DOR-mediated cytoprotection against hypoxia and/or MPP⁺. (A) The PC12 cells transfected with DOR siRNA or PINK1 siRNA together with the cells transfected with NC siRNA were exposed to hypoxia at 1% O₂ for 48 hrs or 1.0mM MPP⁺ for 24 hrs, and then the superoxide fluorescence was detected using MitoSOX Red Mitochondrial Superoxide Indicator. C: control. H: hypoxia. H+U: DOR activation with UFP-512 in hypoxic condition. M: MPP⁺. M+U: DOR was activated using UFP-512 and then exposed to MPP⁺. N=3 in each group. NS: not significant; ^ΔΔp<0.01 vs. M within the same group. Note that the statistical data analyzed by flow cytometer showed that DOR knockdown or PINK1 knockdown greatly interfered with the effects of DOR activation on mitochondrial superoxide especially under MPP⁺ conditions. (B) PC12 cells transfected with DOR siRNA or PINK1 siRNA together with the control were exposed to hypoxia at 1% O₂ for 48 hrs or 1.0mM MPP⁺ for 24 hrs, cytochrome c in the cytosol were extracted and measured using Western blot. N=3 in each group. NS: not significant; ^Δp<0.05, ^ΔΔp<0.01 vs. H or M within the same group. Note that the knockdown of PINK1 or DOR significantly increased the release of cytochrome c from mitochondria to cytosol under hypoxic and/or MPP⁺ insults. The effects of DOR activation on cytochrome c were largely deprived by PINK1 knockdown or DOR knockdown.