Figure 6. DOR activation enhanced mitophagy under normoxic and MPP+ conditions, but not in hypoxia. C: control. CCCP: PC12 cells were treated with 10 μM CCCP for 24 hrs. C+U: cells were treated with DOR agonist UFP-512 in normal conditions. C+U+N: cells were treated with DOR agonist UFP-512 plus DOR antagonist naltrindole under normal conditions. H: hypoxia. H+U: DOR was activated using UFP-512 in hypoxic conditions. H+U+N: DOR was treated with UFP-512 plus naltrindole at the same time in hypoxic conditions. M: MPP+. M+U: DOR was activated using UFP-512 and exposed to MPP+. M+U+N: PC12 cells were treated with UFP-512 plus naltrindole and exposed to MPP+. (A) Quantification of mtDNA was carried out using qPCR under normoxic, hypoxic and MPP+ conditions. N=3 for each group. NS: not significant, *p<0.05, **p<0.01 vs. control. ΔΔp<0.01 vs. M. Note that exposure to CCCP or hypoxia induced significant reduction of mtDNA content in PC12 cell line. The administration of DOR agonist UFP-512 remarkably decreased the mtDNA relative content in the conditions of normoxic and MPP+, whereas no appreciable change was observed under hypoxic conditions. (B) N=3 for each group. NS: not significant, *p<0.05, **p<0.01 vs. control. ΔΔp<0.01 vs. M. Mitophagy was analyzed by measuring the degradation of COXII. Note that CCCP or hypoxic exposure significantly decreased COXII expression. DOR activation by UFP-512 promoted the degradation of COXII under normoxic and MPP+ conditions, whereas showed a minimum effect under hypoxia. (C) N=3 for each group. NS: not significant. Δp<0.05 vs. C or H or M. PC12 cells fluorescent imaging and quantification of co-localization of Parkin/mitochondria were performed in PC12 cell line. Note that exposure to CCCP caused a remarkable increase in co-localization of GRP-Parkin and RFP-mitochondria. The administration of UFP-512 significantly increased the overlap of Parkin/mitochondria under normorxia and MPP+, while showed a minimum effect under hypoxia.