Figure 4.

Regulation of mitochondria by CMA through PARK7. (Ato C) The effect of overexpression of PARK7 on shLamp2A- and MPP⁺-induced mitochondrial dysfunction. SN4741 cells transfected with control GFP or shLamp2a-GFP lentivirus were transfected with the control or PARK7 plasmids, treated with MPP⁺ (50 μM) for 24 h and analyzed (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01). For mitochondrial morphology, cells were stained with MitoTracker Red (10 nM) for 20 min at 33°C. Mitochondrial morphology in living SN4741 cells was analyzed by live-cell imaging (Nikon, C2 Si, Japan) (green, GFP; red, MitoTracker Red; insets represent boxed areas) using ImageJ 1.41 software (A). The right graph shows the form factor of mitochondria, which reflects the complexity and branching, calculated as (perimeter²)/(4π·surface area). For MMP, cells were incubated with TMRE (200 nM) for 20 min at 33°C. Ten thousand cells were assayed for TMRE fluorescence by flow cytometry (B). For ROS levels, cells were incubated with CellROX→ Deep Red Reagent (2.5 μμ). Ten thousand cells were assayed for CellROX→ Deep Red Reagent fluorescence by flow cytometry (C). Scale bar: 50 μm. (Dto F) The effect of downregulation of PARK7 on LAMP2A-induced protection of mitochondria. SN4741 cells transfected with the control or LAMP2A plasmid with or without siRNA oligonucleotides for Park7 were treated with MPP⁺ (50 μM) for 24 h. Mitochondrial functions were assayed as described in (Ato C) (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01). Scale bar: 50 μm. (G and H) The effect of overexpressing PARK7 QE→AA on MMP (G) and ROS (H) under oxidative stress. SN4741 cells were transfected with the indicated plasmids and treated with MPP⁺ for 24 h. MMP and ROS were assayed as described as above. Right graphs show the quantifications (mean ± SEM; n = 3; *, P < 0.05; **, P < 0.01).