Research Paper Volume 12, Issue 24 pp 25035—25059

Figure 7. DOR activation promoted mitophagy under normal conditions and MPP⁺ insults in a PINK1-dependent manner. C: control. C+U: cells were treated with DOR agonist UFP-512 in normal conditions. 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⁺. (A) PC12 cells transfected with PINK1 siRNA were exposed to hypoxia at 1% O₂ for 48 hrs or 1.0mM MPP⁺ for 24 hrs, and then the mtDNA content were measured using qPCR. N=3 in each group. NS: not significant, ^ΔΔp<0.01 vs. C or M within the same group. Note that DOR activation induced down-regulation of mtDNA content was significantly attenuated by PINK1 knockdown both under normoxic and/or MPP⁺ conditions. The application of DOR agonist UFP-512 showed an unappreciable effect on mtDNA content after cells were transfected with PINK1 siRNA under hypoxic condition. (B) COXII degradation was evaluated before and after PINK1 knockdown. N=3 in each group. NS: not significant. p<0.01 vs. C or M within the same group. Note that the administration of DOR agonist UFP-512 down-regulated COXII expression under normoxia and MPP⁺, whereas PINK1 knockdown interfered with DOR mediated COXII degradation. (C) N=3 in each group. NS: not significant, ^Δp<0.05 vs. C or M within the same group. PC12 cells were transfected with negative control siRNA or PINK1 siRNA. Fluorescent imaging and quantification of co-localization of Parkin/mitochondria were performed in PC12 cell line. Note that PINK1 knockdown seriously interfered with the co-localization of GRP-Parkin and RFP-mitochondria induced by DOR activation under normal and MPP⁺ conditions. The overlapping of Parkin/mitochondria showed no appreciable difference between the cells transfected with NC siRNA and the cells transfected with PINK1 siRNA under hypoxic condition.