Fv3000

In mitochondrial morphological analysis, 5–10 z-stack images (1-μm steps) of pMT-mKO1 plasmid-transfected cells were acquired using a confocal microscopy system (FV3000, Olympus). Deconvolution was applied to maximum projection images to clarify the mitochondrial contours. Mitochondrial length was defined as the longitudinal axial length of each mitochondrial fragment, which were traced with cellSens life science imaging software (Olympus). Mitochondrial length was calculated as the mean length of mitochondria within a single cell.
In ER morphological analysis, mCherry-Sec61B-expressing cells were kept in a humidified atmosphere at 37°C using a Stage Top incubator (Tokai-hit). Live cell images were acquired using the confocal microscopy system (FV3000, Olympus). The standard deviation (SD) and mean of the fluorescence intensity were obtained using line-scan analysis by the FV31S-SW (Olympus) in a randomly selected cell region. The ER morphology was defined as reticular when the coefficient of variation (CV) scores (SD/mean) were >0.2.

To analyse the formation of RAD52 GFP foci, WT and ΔQ cells were grown until the exponential phase and treated with 0.03% MMS and 15 mM GO for 1 hr. Subsequently, the A₆₀₀=0.5 OD cells were harvested and washed with 1 X PBS before spreading over 2% agarose pads. The number of foci were quantified and plotted.
The localization of yeast DJ-1 members was performed by genomically tagging with GFP at the carboxy terminus using primers P12-P14. Consequently, the cells were transformed with a pRS415_TEF vector expressing MTS-mCherry to decorate mitochondria (Bankapalli et al., 2015 (link)). The transformed strains grown until the mid-exponential phase were harvested (A₆₀₀=0.5) and incubated with water or 15 mM GO for 2 hr. Parallelly, the cells were treated with water or 10 mM MG for 2 hr and harvested. The cell pellet was washed with 1 X PBS and processed for imaging. All the images were acquired in confocal microscopy (Olympus FV3000) with a 10 µm scale bar.
For mitochondrial DNA visualization, selected strains were grown till the mid-log phase and treated with 15 mM GO for 3 hr. Later, cells were harvested and stained with 10 μM SYTO 18 for 15 min at 30 °C, followed by visualization under confocal microscopy (Olympus FV3000). Images contain 10 µm scale bar.

For calculating hu-α-syn pathology and hu-α-syn/Iba-1-positive cell in vivo, stained brain slices (30 μm) z-stack pictures (×40 magnification) were captured by confocal fluorescence microscopy (FV3000; Olympus Life Science) at multiple areas of the STR and CTX. For quantification of hu-α-syn pathology, three or four brain slices were used for each brain, and positive signals were counted using ImageJ after setting the threshold. More specifically, hu-α-syn signals from 5 days after the injection were calculated using integrated density, whereas hu-α-syn signals from 1 month after the injection were calculated with the analyzed particles. Hu-α-syn inside microglia was manually counted in a z-stack picture.
Confocal microscopy (FV3000; Olympus Life Science) was used to capture z-stack images at ×40 magnification to calculate hu-α-syn deposits in microglia and astrocytes in vitro. the ImageJ software was used to quantify hu-α-syn-, Iba-1-, and GFAP-positive areas to quantify the hu-α-syn clearance in microglia and astrocytes. Colocalized areas of Iba-1 and hu-α-syn or GFAP and hu-α-syn were quantified to limit the analysis to internalized hu-α-syn only.

The StubRFP-SensGFP-LC3 assay was performed according to the manufacturer’s instructions (GeneChem, Shanghai, China). Briefly, U87 cells (5 × 10⁴ cells/mL) seeded in a culture dish were incubated with StubRFP-SensGFP-LC3B lentivirus (MOI = 5 × 10⁶ TU/mL). The medium was changed after 12 h of incubation. After infection for 72 h, the cells were treated with TMZ at the indicated dosage for the indicated time and then treated with MitoTracker deep red (Invitrogen company, Eugene, OR). Finally, the cells were observed under a confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan).
Then, U87 cells seeded in a culture dish were incubated in the presence or absence of TMZ at the indicated dosage for the indicated times before they were treated with 100 nmol/L MitoTracker red (Invitrogen Company, Eugene, OR) for 30 min at 37 °C before fixation in ethanol. After the nonspecific antibody binding sites were blocked, the cells were incubated with anti-BNIP3 (1:100) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) for 1 h. Finally, the cells were visualized under a laser scanning confocal microscope (Olympus FV3000, Tokyo, Japan).

Cells were washed three times with ×1 PBS and then fixed with 4% paraformaldehyde (PFA) for 12 min. After being penetrated by Triton X-100 (0.25%–0.5%) for 5 min, the cells were blocked by bovine serum albumin (BSA, 5%) for 2 h. Cells were incubated with antibodies, e.g., phalloidin (FITC, A12379, Thermo) and vinculin (ab196579, Abcam, Cambridge, UK) antibodies. Then, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Dapi, D9542, Sigma, St. Louis, MO) was applied to stain the nuclei for 10 min at 10 μg·mL⁻¹. The immunofluorescence images of cell samples were observed through a confocal laser scanning microscope (FV3000, Olympus, Tokyo, Japan and A1R MP+, Nikon, Tokyo, Japan, and parameter: 40, Nikon Microsystems original image: 1 024 × 1 024). The images presented in the figures are representative of a projection of all slices based on at least three independent experiments (n = 3). For the quantification of immunofluorescence intensity, we used the connected software in FV3000, Olympus and A1R MP+, Nikon.