Triton x 100

LX2 cells were cultured on chambered borosilicate coverglasses (Thermo Scientific, Rockford, IL, USA), treated, and stained with specific fluorochromes to assess colocalization between LC3 and lipid droplets. Following treatments, cells were fixed for 15 min with 100% methanol (VWR Chemicals, Radnor, PA, USA) at −20 °C and were then blocked for 1 h at room temperature in PBS containing 5% goat serum and 0.3% Triton™ X-100 (Sigma Aldrich, Steinheim, Germany). Cells were incubated for 18 h in PBS with 1% BSA and 0.3% Triton X-100 containing LC3 XP Rabbit mAb antibody (4599, Cell Signaling Technology, Danvers, MA, USA, 1:400) at 4 °C and washed with PBS. Samples were then incubated for 1 h with a secondary antibody conjugated with Alexa 488 Goat Anti-rabbit (A-11008, 1:500, Molecular Probes, Life Technologies, Madison, WI, USA) at room temperature, and Hoechst 33342 (3 µM, Sigma Aldrich, Steinheim, Germany) was added for the last 30 min. After washing cells with PBS, they were incubated for 10 min in HBSS containing 0.5 µM Nile Red. Fluorescence was detected with a Leica fluorescence confocal microscope (Leica microsystems CMS GmbH, Mannheim, Germany), and the Colocalization Colormap Plugin was used to calculate the Correlation Index (Icorr).

6 male B6 wild-type mice were raised under specific pathogen-free environment for 1 year. AAVs carried miRNA (Obio Technology, China) were injected to the substantia nigra pars compacta (SNc) of the mice brain through stereotaxic surgery. The coordinate axis parameter of SNc is ±1.25 mm in M/L, −3.16 mm in A/P, −4.25 mm in D/V. After 3 weeks, the MPTP (Sigma, United States) was administrated in these mice with injection every 2 h for four doses in total over an 8 h period (20 mg/kg per dose × 4). One-day interval later, these mice were sacrificed and their brains were removed. The brain tissues were fixed with 10% paraformaldehyde (Cell Signaling Technology, United States) for 1 day at room temperature, followed by dehydration in 30% sucrose for 2 days at 4°C and embedding with O.C.T.Compound (Solarbio, United States) at −80°C. The brain tissues were cut to 20 μm thick slices with the freezing microtome (Leica, Germany). The slices were blocked with 5% BSA powder (Proliant, United States) dissolved in PBS containing 0.3% Triton X-100 (Cell Signaling Technology, United States) for 1 h followed by incubation of rabbit tyrosine hydroxylase (TH) primary antibody (Abcam, United States, 1:200) overnight at 4°C and then with Fluor 594 (red) secondary antibody (ThermoFisher, United States) at 37°C for 30 min. Images were taken by Zeiss LSM 880 confocal microscope.