D141400

Cells were plated on 35 mm multi-well glass bottom dishes (Matsunami Glass, D141400). Transfection was performed with X-tremeGENE 9 (Roche) according to the manufacturer’s instructions; 16–20 hr after transfection, the growth medium was replaced with phenol red-free Dulbecco’s modified Eagle’s medium (glucose 4.5 g/l) supplemented with 10% fetal bovine serum and 2 mM glutamine. During image acquisition, cells were incubated on a Tokai Hit stage top incubator at 37°C in a humidified 5% CO₂/95% air atmosphere. Images were acquired with an LSM880 confocal microscope with Airyscan (Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective lens. Image acquisition was performed using ZEN software (black edition 2.3). Super-resolution images were obtained by subjecting raw images to the Airyscan processing program. Fiji software was used for image presentation.

Intracellular delivery of PIP₃ was performed using the PIP₃ Shuttle PIP™ Kit (P-9039, Echelon Biosciences), with slight modifications to the manufacturer’s protocol. Briefly, HeLa cells stably overexpressing αSyn-mRFP were seeded on a 4-well glass bottom dish (Matsunami, D141400) and incubated overnight at 37 °C. Shuttle PIP carrier 2 (Histone H1) and Bodipy^®-FL-PIP₃ were incubated in a 0.2 ml tube in a 1:1 molar ratio for 10 min at room temperature. The complex was diluted with Opti-MEM and added to media covering HeLa cells with a final carrier and PIP₃ concentration of 5 µM. The following day, the dye-containing media was removed, and cells were washed with phosphate-buffered saline (PBS) before live imaging using SpinSR10 (Olympus) or fixation with 4% paraformaldehyde (PFA) for immunostaining. Confocal images were taken randomly across the entire well at 60× magnification, and the percentage of αSyn puncta-positive cells was quantified. For transient gene overexpression, HeLa cells were seeded and transfected with pcDNA-αSyn-mRFP 24 h prior to delivery of PIP₃ using Fugene HD transfection reagent (E2311, Promega Corporation), according to the manufacturer’s protocol. For live imaging of lysosomes, cells were incubated with 300 nM LysoTracker™ Blue DND-22 (L7525, Invitrogen) for 1 h and washed three times with PBS before observation.

Hippocampal cells were dissociated from E16.5 mouse embryos by brief trypsinization (0.25% trypsin for 10 min at 37°C) and trituration through a fire-polished Pasteur pipette, and were plated onto a 9.5-mm multi-well glass-bottom dish (D141400, Matsunami, Japan) at 550–600 cells/mm². On DIV 20–21, the culture medium was replaced with a calcium-indicator loading solution consisting of 0.0005% Oregon Green BAPTA-1AM, 0.01% Pluronic F-127, and 0.005% Cremophor EL in aCSF (127 mM NaCl, 26 mM NaHCO₃, 1.5 mM KCl, 1.24 mM KH₂PO₄, 1.4 mM MgSO₄, 2.4 mM CaCl₂, and 10 mM glucose). The cells were incubated for 1 h in a 37°C humidified incubator and then washed several times with aCSF to remove the loading solution. Live calcium imaging was performed with an LSM780 confocal laser scanning microscope equipped with the Incubation System XL (Zeiss, Germany). A 105.9 μm × 105.9 μm visual field was imaged for 2 min with an imaging interval of 100 ms. Data derived from the Pcdhαβγ^+/+:TG^taf7 and Pcdhαβγ^del/+:TG^taf7 genotypes were used as controls.

A total of 2.0 × 10⁴ cells per well were seeded in multiwell glass bottom dishes (D141400, MATSUNAMI, Osaka, Japan). mtDNA in cells was stained with SYBR Green I at a 1:600,000 dilution for 30 min and washed with PBS four times as previously described [31 (link)]. Then, the MMP was determined via staining with MitoTracker Orange (M7510, Invitrogen, California, UA) for 10 min followed by two washes with PBS. Fluorescence immunostaining was evaluated by a Confocal Laser Scanning Microscope FV3000 (Olympus, Tokyo, Japan). This fluorescence was quantified by ImageJ v1.53 software, which is an open source and public domain image processing software.

The J774A.1 was seeded into a multiwell glass-bottom dish (D141400, Matsunami Glass Ind., Ltd., Osaka, Japan) at 8.0 × 10⁴ cells per well and cultured for 24 h in DMEM containing 10% FBS in a humidified 5% CO₂ at 37°C. Infection of R. equi and cell culture were performed under the same conditions as above. The luminescence signals in the cells were detected using an IX83 microscope (Olympus, Tokyo, Japan).