Research Paper Volume 12, Issue 24 pp 25035—25059

Figure 3. Beneficial effects of DOR activation on mitochondria under hypoxic and/or MPP⁺ insults were partially blocked by PINK1 knockdown or DOR knockdown. (A) BC: blank control. PC12 cells were merely transfected with lipofectamine 2000. NC: negative control. PC12 cells were transfected with negative control siRNA. DOR siRNA: PC12 cells were tranfected with two kinds of DOR siRNA 1 and 2. PINK1 siRNA: PC12 cells were transfected with PINK1 siRNA. N=3 for each group. ^*p<0.05, ^**p<0.01 vs. BC. Note that the expression intensity of DOR was significantly interfered by DOR siRNA 2, and PINK1 expression was significantly reduced by PINK1 siRNA transfection. (B) PC12 cells were exposed to 1% O₂ for 48 hrs or 1.0mM MPP⁺ for 24 hrs. C: control. H: hypoxia. H+U: DOR was activated using UFP-512 in hypoxic conditions. M: MPP⁺. M+U: DOR was activated using UFP-512 and exposed to MPP⁺. N=3 in each group. ^ΔΔp<0.01 vs. H or M; NS: not significant. Note that DOR knockdown or PINK1 knockdown caused a significant decrease in ATP production both under the conditions of hypoxia and/or MPP⁺. Treat cells with DOR agonist failed to restore the capacity of mitochondria in ATP generation after cells were transfected with DOR siRNA or PINK1 siRNA. (C) PC12 cells were exposed to 1%O₂ for 48 hrs or 1.0 mM MPP⁺ for 24 hrs, the mitochondrial membrane potential was measured using TMRM. N=3 in each group. NS: not significant; ^Δp<0.05 vs. H; ^ΔΔp<0.01 vs. M. Note that the knockdown of DOR or PINK1 significantly blocked the effects of DOR activation on attenuating mitochondrial membrane potential collapse both under hypoxic and/or MPP⁺ insults.