Cell model construction and grouping
Rat adrenal pheochromocytoma cells PC-12 were purchased from Wuhan Prosaic Technology Co. The cell line was cultured in 1640+10% inactivated fetal bovine serum medium, which contains 100 U/mL penicillin and 100 U/mL streptomycin. Placed in a constant temperature incubator containing 5% CO2 at 37℃, the medium was changed every 2 days, and when the cell growth reached 80%-90%, passaging, plate spreading and sample collection were performed. Logarithmically grown cells were inoculated in 96-well plates and cultured for 24 h. MPTP (500 µM) was added and treated for 24 h to induce an in vitro PD model.
The cells in the experiment were divided into four groups: OE-NC; OE-PCBP1; OE-NC+MPTP (500µM); OE-PCBP1+MPTP (500µM).
RT-qPCR
Total RNA was extracted from each group of cells using the chloroform isopropanol method, and the precipitate was dissolved in an appropriate amount of DEPC water. The total RNA concentration (μg/μl)=OD260×40×10-3 was calculated by measuring OD260, OD280 and OD260/OD280 values with a microspectrophotometer. cDNA was reverse transcribed according to the instructions of the reverse transcription kit and then detected by fluorescence qPCR. The primer sequences were as follows: GAPDH, 5'-ACTCCCATTTCCACCTTTG-3' (forward) and 5'-CCCTGTTGCTGTAGCCATATT-3' (reverse). The relative expression of each group of genes was calculated using the 2ΔΔCT method.
Protein blot analysis
Frozen tissue samples were lysed with RIPA lysate. Protein concentration was determined by BCA method after lysis on ice for 30 min, sonication in an ice bath for 3 min, and centrifugation at 4°C for 10 min at 12000 rpm. The protein blotting procedure is described elsewhere (Lee et al., 2011) [9]. Briefly, protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After closure in 5% TBST, the primary antibody was diluted with 1% BSA / PBST at the recommended dilution ratio and the membrane was closed overnight at 4°C with a hybridization bag. After 3 washes in PBST, the membrane is incubated with the secondary antibody for 1 hour at room temperature. The secondary antibody is completely removed by four washes in PBST. These membranes were developed using a chemiluminescence assay system (Roche) and exposed to Kodak film. Blots were quantified by the Image J program.
Cell viability assay
Cell viability was checked by the CCK8 method using a cell counting kit (CK04 Tohin Chemical). Briefly, at the end of the treatment, the cell culture medium was removed and 10 μ l of working solution was added to each well. Incubate for 2 h at 37°C in the dark. Using the enzyme marker, measure the absorbance at 450 nm.
Apoptosis assay
Apoptosis was detected using the Annexin V-FITC/PI Apoptosis Kit (Model: 40302ES60, NextSaint Biotechnology). Cells were collected 48h after plasmid transfection, washed with PBS, centrifuged and supernatant discarded, 100μl Annexin Binding Buffer was added and cells were resuspended and stained with 5μl FITC and 10μl PI for 15min at room temperature and protected from light, 200μl Annexin Binding Buffer was added before the machine. Finally, apoptosis was analyzed by flow cytometry (model: NovoCyte, ACEA).
Mitochondrial membrane potential assay
JC-1: Mitochondrial membrane potential (MMP) was analyzed using the JC-1 staining kit according to the manufacturer's instructions. Briefly, 1 ml of JC-1 (1X) was added to each well and incubated for 20 min at 37°C in the dark. After incubation, cells were washed 3 times with cold PBS buffer. absorbance at 590 nm represents high membrane potential (red) and absorbance at 530 nm indicates low membrane potential (green). Images were collected using a fluorescence microscope (IX73, Olympus, Japan). Fluorescence intensity was quantified by Image J software. The average fluorescence intensity was calculated from 10 regularly distributed regions in each image.
ROS: ROS results were analyzed using the DCFH-DA kit according to the manufacturer's instructions. Briefly, DCFH-DA at a final concentration of 10umol/L was added to each well and incubated for 20 minutes at 37°C in the dark. After incubation, the cells were washed three times with serum-free medium to adequately remove DCFH-DA that had not yet entered the cells, and the appropriate amount of buffer was added to the machine and assayed.