Live-cell imaging

Wash cells 1x with OptiMEM (Gibco).

Incubate cells with 100nM MitoTracker red CMH2Xros (Thermo Fisher Scientific) in OptiMEM at 37 °C for 30 min

Wash cells with OptiMEM once, and keep cells in the same medium for imaging

Images were acquired using a Leica TCS SP8 confocal microscope (Leica, Germany) with a 100×/1.4 numerical aperture oil-immersion objective.

Analyze images using Diffraction PSF 3D and DeconvolutionLab2 plugins in Fiji-ImageJ version 2.3.0/1.53q (https://fiji.sc; RRID:SCR_002285)

Treat iPSC-derived neurons with 0.25 μM Alexa Fluor 594-labeled PFFs (594-PFF) with or without cotreatment with 1 μM CDDO-Me (Cayman Chemical)

After 24 h, incubate cells with 100 nM MitoTracker Green (Invitrogen, MA, USA) in a neuronal medium for 30 min at 37 °C

Use ACellBrite™ Steady 488 Membrane Staining Kit (Biotium) to visualize cell membranes following the manufacturer's instructions

Images were acquired using a Leica TCS SP8 confocal microscope with a 63 × /1.4 numerical aperture oil-immersion objective

Acquire Z-stacks for the calculation of the PFF particle area and fluorescence intensity

For each condition, 5-8 images were acquired from at least four independent experiments

For the quantification of colocalization and image processing, images were analyzed using the “Analyze particles” and “EzColocalization” plugins in Fiji-ImageJ