Cell culture and drug treatment
The human neuroblastoma cell line SH-SY5Y was obtained from Sun Ye San University (Guangzhou, China) and cultured in DMEM/H medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% glutamine in a humidified incubator with 5% CO2 at 37°C. We changed the culture medium every two days and subcultured the cells when the density reached 80%. (1) To study the effect of MPP+ and/or ART on cell viability, we treated the cells with the indicated concentrations of MPP+ and/or ART for 24 h and performed a CCK-8 assay. (2) To study the protective effect of ART on MPP+-treated SH-SY5Y cells, we treated the cells with PBS, ART, MPP+, or ART+MPP+ for 24 h. The concentration of ART was 20 μM, and the concentration of MPP+ was 1 mM. (3) To study changes in autophagy, we treated the cells with PBS, ART, MPP+, ART+MPP+ or 3-MA+MPP+ for 24 h. The concentration of ART was 20 μM, the concentration of MPP+ was 1 mM, and the concentration of 3-MA was 5 mM. MPP+ was purchased from Sigma (CAS No. 36913-39-0, USA). ART was purchased from DASF (CAS No. 71963-77-4, Nanjing, China). The autophagy inhibitor 3-methyladenine (3-MA) was purchased from Sigma (M9281, USA).
Cell viability
Cell viability was measured by CCK-8 assay (Solarbio, CA1210, China), according to the manufacturer’s instructions. Briefly, 5×103 SH-SY5Y cells/well were plated in 96-well plates and incubated for 24 h. Then, the cells were treated with MPP+ and/or ART for another 24 h. Then, 100 μl culture medium containing 10 mM CCK-8 was added to each well and incubated at 37°C for 2 h. The absorbance was measured at 450 nm with a multimode microplate reader (EnSpire, PerkinElmer, Singapore). The cell viability of the control group was set to 100%, and the cell viability of the other groups was compared with that of the control group.
Intracellular Reactive Oxygen Species (ROS) detection
The levels of intracellular ROS were measured by a ROS assay kit (Beyotime Biotechnology, S0033, China), according to the manufacturer’s instructions. After drug treatment, the SH-SY5Y cells were incubated with serum-free medium containing 10 μM DCFH-DA at 37°C for 20 min and then washed with serum-free culture medium three times. After adding 1 ml fresh medium to each well, the fluorescence was observed using the GFP channel of an inverted fluorescence microscope (Nikon, Japan). The fluorescence intensity was analyzed by ImageJ.
Superoxide dismutase (SOD) activity assay
The intracellular SOD activity was measured with a total SOD activity detection kit (Beyotime Biotechnology, S0101, China), according to the manufacturer’s instructions. After drug treatment, the cells were washed once with PBS and collected by centrifugation. The cells were fully lysed in SOD sample preparation solution, and the supernatants were collected at 12,000 × g at 4°C for 5 min. The absorbance was detected at 450 nm and 600 nm (reference wavelength) with a multimode microplate reader, and the SOD activity was calculated.
Glutathione (GSH) assay
The content of GSH was measured with a glutathione assay kit (Beyotime Biotechnology, S0052, China), according to the manufacturer’s instructions. After drug treatment, the cells were washed once with PBS and collected by centrifugation. After the samples and standards had been prepared, the absorbance was detected at 412 nm. We generated a standard curve and calculated the contents of GSH in the samples based on this standard curve.
Malondialdehyde (MDA) assay
The intracellular MDA levels were measured with an MDA detection kit (Beyotime Biotechnology, S0131, China), according to the manufacturer’s instructions. After drug treatment, the cells were collected. The cells were lysed, and the supernatants were collected at 10,000 × g for 10 min. After the samples and standards had been prepared, the absorbance was detected at 532 nm and 450 nm (reference wavelength). The MDA contents in the samples were quantified based on the standard curve.
Mitochondrial membrane potential (MMP) assay
To monitor the mitochondrial integrity, a mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotechnology, C2006, China) was used, according to the manufacturer’s instructions. Briefly, after drug treatment, the SH-SY5Y cells were incubated with JC-1 working solution at 37°C for 20 min and washed twice with JC-1 buffer. Red fluorescence and green fluorescence were observed with the GFP channel and TRITC channel of an inverted fluorescence microscope (Nikon, Japan). The fluorescence intensity was analyzed by ImageJ.
Flow cytometry assay
We used an Annexin V-FITC cell apoptosis detection kit (Beyotime Biotechnology, C1062M, China) to detect cell apoptosis, according to the manufacturer’s instructions. After drug treatment, the cells were harvested and resuspended in PBS. A total of 50,000 to 100,000 cells were collected and resuspended in 195 μl Annexin V-FITC binding buffer, and then, 5 μl Annexin V-FITC and 10 μl PI were added with gentle mixing. The cells were incubated at room temperature for 20 min, and then, the fluorescence was detected by flow cytometry (Accuri C6, BD).
Western blot assay
Cell lysates were prepared, and the protein concentrations were quantified with a BCA protein assay kit (CWBIO, CW2011S, China). Samples with equal amounts of proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated at 4°C overnight with primary antibodies (1:1000) against cleaved caspase-3 (Cell Signaling Technology, Aps175, USA), caspase-3 (Abcam, ab90437, UK), LC3 (Abcam, ab128025, UK), Beclin1 (BD, 612113, USA), P62 (Abcam, ab56416, UK) or β-actin (1:5000; CWBIO, CW0096M, China). The next day, the membranes were washed with TBST three times and incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (CWBIO, 1:5000) at room temperature for 1 h. The blots were visualized with a cECL Western blot kit (CWBIO, CW0049M, China), and the images were analyzed by ImageJ.
Immunofluorescence Staining
SH-SY5Y cells were seeded on slides in 12-well culture plates. After drug treatment, the cells were fixed with ice-cold methanol for 5 min, blocked with 1% bovine serum albumin (BSA) for 30 min, and then incubated with primary antibodies against LC3 (1:200) and P62 (1:100) at 4°C overnight. The next day, the slides were incubated with Alexa Fluor 488 (Bioss Antibodies, bs-0295G-AF488, China) or Alexa Fluor 555 (Bioss Antibodies, bs-0296G-AF555, China) secondary antibodies (1:500) at 37°C for 1 h and incubated with DAPI (Boster, AR1177) for 5 min. Fluorescence was observed with a fluorescence microscope (Olympus, BX53).
Statistical Analysis
The results are expressed as the mean±standard deviation (mean±SD). Comparisons among multiple groups were performed with one-way ANOVA followed by Tukey’s post hoc test, and P <0.05 was considered statistically significant.