1. Cell culture, differentiation, hypoxia, glucose deficiency model establishment, and drug administration treatment
PC12 cells have been used in the study of spinal cord injury. The PC12 cell line is derived from neuron-like rat adrenal pheochromocytoma cells [30,31]. The cells utilized in this study were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 1 × 106 cells were cultured on each 10cm culture dish that was pre-treated with poly-L-lysine at room temperature for one hour then PBS washed. The cells were cultured in RPMI-1640 complete culture medium (Gibco, CA,USA) with 10% horse serum (Gibco, CA, USA, New Zealand origin), 5% fetal bovine serum (FBS; Gibco, CA,USA, Brazil origin), and 1% penicillin / streptomycin (Gibco, CA, USA) in a 37 °C, 5% carbon dioxide incubator.
To induce the differentiation to PC12, we used RPMI medium with 50 ng/ml nerve growth factor (Sangon Biotech, Shanghai, China), 10% fetal bovine serum, and 1% penicillin / streptomycin after allowing the cells to adhere for 24 h. The medium was changed every 2 days until day 5 when we observed a marked increase in axonal growth; at this stage, the differentiation was completed and the cells were ready for experimentation. The OGD model can induce the death and apoptosis of PC12 cells differentiated by NGF to simulate ischemic injury of the living spinal cord. The medium in the cultured PC12 cells after differentiation was aspirated and washed gently with PBS three times before adding Hank's Balanced Salt Solutions medium (Gibco, CA, USA) without glucose and transferring to a preset triple gas incubator (Thermo, PA, USA) containing 1% O2, 5 % CO2, and 95% N2 hypoxia. To investigate whether LSD1 mediates autophagy and apoptosis in PC12 cells through the PI3K/Akt/mTOR signaling pathway, 50 uM of GSK-LSD1 and 10 uM of IGF-1 (20 mg/L, Merck Millipore, Millipore, Billerica, MA, USA) were added to the culture media for 48h.
2. Cell viability assay to screen drug concentrations
The middle wells of a 96-well plate were selected to avoid the effects of medium evaporation which are more likely in the outer wells. Then, PC12 cells were added at 1.5×104 cells/well. We also included a blank group (without cells to eliminate the background, a control group (only complete medium was added to induce differentiation), and a dosing group (according to the gradient), and three replicate wells were set for each group. After the cells adhered, we added GSK-LSD1 (0, 25, 50, 100, 200 μM) to the media and incubated the cells in a 5% CO2 incubator at 37°C for a further 48 h. To determine viability, we replaced the medium with 100 μl of fresh medium containing 10 μl of CCK8 solution (Dojindo Laboratories, Kyushu, Japan) to each well, incubated for 2 h in a 37°C incubator, and then incubated for 2 h using an enzyme-linked immunoassay (BioTek, VT, USA). Viability was determined by measuring the absorbance values at a 450 nm wavelength. The cell survival rate (%) was calculated as follows: (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%.
3. LSD1 gene knockdown
We used lentiviral transfection to knockdown the LSD1 gene in PC12 cells. The constructed lentivirus (MOI=20) and 6μg/ml of Polybrene ( G e n e -Pharma, Shanghai, China) were added to the cells for 24 h. Transfected cells were selected by using 5µg/ml puromycin for 7 days until we determined 0% viability in the control group; then, we used 2.5µg/ml puromycin for maintenance. After selection, cell protein and RNA were collected for western blot and qPCR, respectively, to verify knockdown efficiency. PC12 cells showing a good knockdown were used in subsequent experiments.
4. Transmission electron microscopy of autophagic vesicles
The treated PC12 cells were fixed with 2.5% glutaraldehyde for 2 h and then scraped off with a cell scrapers and collected in 15 ml centrifuge tubes. After curing in the oven, the sections were sectioned on an LKB-1 slicer, stained with 4% uranyl acetate for 20 min, then lead citrate for 5 min. The sections were imaged by using a transmission electron microscope (Hitachi, Tokyo, Japan).
5. Flow cytometric quantification of apoptosis and necrosis in PC12n cells
Each group of cells was gently digested with EDTA-free trypsin, then neutralized with complete differentiation medium, and centrifuged at 1200 rpm/min for 5 min. The pellet was washed twice with 4°C PBS solution. Then, 5 μl of Annexin V-FITC (BD Bioscience, CA, USA) was added to each tube and incubated for 15 min at room temperature in the dark. Finally, 5 μl of PI (BD Bioscience, CA,USA) was added to each tube and the signal was acquired by a flow cytometer (BD Bioscience, CA,USA) after 5 min. The percentages of early apoptotic cells (AV+/PI-), late apoptotic cells (AV+/PI+), and necrotic (AV-/PI+) cells in the sample were determined by using BD FACSDiva 7.0 software.
6. Immunohistological assessment of cell apoptosis
The treated cells were washed with PBS then fixed and permeabilized. We then used a terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay working solution to the cells, incubated for 60 min at 37°C in a dark temperature chamber. The nuclei were stained with DAPI, blocked with a solution containing a fluorescence quencher, and images were captured using an inverted fluorescence microscope (Leica, Heidelberg, Germany).
7. Immunohistological assessment of cell autophagy
Cells were washed with medium and PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 (Solarbio, Beijing, China), washed and blotted with PBS, and then fixed with rabbit monoclonal anti-LAMP1 primary antibody (Abcam, Cambridgeshire, England) and mouse monoclonal LC3 primary antibody (Abcam, Cambridge-shire, England) diluted 1:50 in 1% BSA. A small circle was drawn on each Petri dish with a hydrophobic histochemical and 50 μL of diluted primary antibody was added dropwise to the circle. The dish was then incubated at 4°C overnight. The next day, the cells were incubated in fluorescent secondary antibody, DAPI stained, blocked, and images were acquired. quantification was performe by using ImageJ software (NIH, Bethesda, MD).
8. Western blot assessment of protein
Total protein was extracted from the cells and the concentrations were determined using a BCA kit (Boster Biotech, Wuhan, China). The proteins were transferred to PVDF membranes via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were blocked with 5% skim milk powder for 2 h at room temperature and incubated with LSD1/KDM1A (Abcam, Cambridgeshire, England), p62 (Cell Signal Technology, MA, USA), Beclin1 (Cell Signal Technology, MA, USA), ATG5 (Cell Signal Technology, MA, USA), ATG7 (Abcam, Cambridgeshire, England), LC3B (Abcam, Cambridgeshire, England), Bax (Cell Signal Technology, MA, USA), Bcl2 (Abcam, Cambridgeshire, England), Caspase3 (Abcam, Cambridgeshire, England), Caspase3 (Abcam, Cambridgeshire, England) and β-actin (Cell Signal Technology, MA, USA) at 4°C for 12 h at a dilution of 1:1000. We then incubated with peroxidase-labeled secondary antibody (1:5000, Dingguo, Beijing, China) at room temperature for 1 h and then with incubated with a chemiluminescent agent (Beyotime Biotech, Hangzhou, China) to show the immunolabeled bands. Protein levels were determined by quantifying band densities using ImageJ software (NIH, Bethesda, MD).
9. Real-time quantitative PCR mRNA quantification
The cells were washed with PBS and the liquid was aspirated. 1mL of Trizol (Thermo, PA, USA) was added to each well; after sufficient lysis, we added 200 ul of chloroform to each well. The supernatant was discarded and 1ml of 75% anhydrous ethanol was added, washed thoroughly, centrifuged, then the pellet was dried. The RNA precipitate was dissolved with DEPC water, and the concentration was measured and reverse transcribed to a cDNA template. The qPCR primers were designed according to the sequences of the LSD1 and β-actin genes. The test was conducted according to the Sybr green kit requirements using the following cycle: pre-denaturation at 55°C for 2 minutes and 1 cycle, followed by denaturation at 95°C for 10 minutes and 1 cycle, denaturation at 95°C for 15 seconds, and then annealing extension at 60°C for 60 seconds for 40 cycles. The amplification curve and melting curve were confirmed at the end of the reaction, and the Ct values were recorded.
10. Immunoprecipitation (co-IP) assay to verify protein interactions
Total protein was measured using a BCA kit (Boster Biotech, Wuhan, China) after extraction. Each sample was adjusted to 1-2ug total protein/μl and divided into three equal aliquots. Two aliquots were incubated with LSD1 or P-PI3K and IgG primary antibodies on a shaker at 4℃ overnight. The next day, magnetic beads were added to 1.5mL Eppendorf tubes and washed twice with a rinse solution. The pre-treated beads were added to the pre-prepared protein sample and placed on a shaker at 4°C overnight. The supernatant was discarded, and rinsed twice with a rinse solution. The beads were added to 50 ul of loading buffer and denatured by boiling in water for 5-10 min. The beads were discarded and the loading buffer was used for subsequent detection by Western blotting.
11. Quantification of cell proliferation
Cells were grown in 96-well plates at 1.5 × 104 per well in 3 groups: control, LSD1 knockout, and LSD1 knockout + IGF-1. We used three replicates per group and five 96-well plates were grown separately, one per study timepoint. OGD treatment was performed over 0, 3, 6, 9, and 12 hours. The plates were pre-cultured in an incubator for 24 h (37°C, 5% C02). After cell treatment, 10ul CCK8 solution (Dojindo Laboratories, Kyushu, Japan) was added to each well and incubated in an incubator protected from light for 1-4 h. The absorbance at 450 nm was measured using an enzyme marker (BioTek, VT, USA).