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A
E3 ligase‐deficient Parkin mutants (T240R and T415N) have no effect on PA accumulation (green) on depolarized, Parkin‐positive mitochondria. The expression of WT and Parkin mutants was determined by Immunoblotting. Scale bar = 25 μM, and zoom is 3×.
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B
DAG accumulation depends upon intact Parkin E3 ligase activity. Scale bar = 25 μm, and zoom is 3×.
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C
Quantification of imaging experiments shown in (A) and (B). Bars represent the mean with SEM; two‐way ANOVA analysis was performed for statistical analysis (***P < 0.001). n = 3 biological replicates.
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D
Hela cells were transfected with control or optineurin‐ and NDP52‐ siRNAs and expression plasmids of FLAG‐Parkin and YFP‐DAGR. These cells were treated with DMSO or 10 μM CCCP for 5.5 h, as indicated. Note that OPTN/NDP52 double knockdown prevented mitochondrial YFP‐DAGR accumulation. (Scale bar = 25 μM and zoom is 3×).
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E
Hela OPTN/NDP52 knockdown cells were transfected with a siRNA‐resistant wild‐type or ubiquitin‐binding deficient E478G OPTN mutant, as indicated. The percentage of cells with mitochondrial YFP‐DAGR following CCCP treatment was quantified. Asterisks indicate statistical significance by one‐way ANOVA (**P < 0.01, ***P < 0.001) from three biological independent experiments. The bars indicate mean ± SEM. Bottom panel: Expression of OPTN, OPTN E478, and NDP52 was determined by immunoblotting.
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F, G
Hela OPTN/NDP52 knockdown cells were incubated with 1,2‐Dipalmitoyl‐sn‐glycerol (DPG) followed by CCCP treatment for 18 h. Mitophagy efficiency was assessed by TOM20 staining and quantified in (G) (n = 2 biological replicates). Note that DPG significantly restored mitophagy in OPTN/NDP52 knockdown cells. Scale bar = 10 μM.
