Culture of the C17.2 cells and establishment model of OGD/R
The C17.2-NSCs was purchased from the American Type Culture Collection (Rockville, MD, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, USA) with 10% (v/v) fetal bovine serum (FBS; Gibco, Carlsbad, USA) in a humidified incubator with 5% CO2 and 95% air at 37°C. In the Oxygen Glucose deprivation/reperfusion model (OGD/R), cells were washed three times with sterilized PBS and the medium was replaced with sugar-free medium culturing in O2/CO2 (1% / 5%) three-gas incubator. After 6 h, the cells medium was replaced with DMEM containing 10% FBS for another 24 h in a humidified incubator with 5% CO2 and 95% air at 37°C.
Lentiviral-mediated KAP1 mutations and plasmid transfection
Lentiviral-mediated KAP1 (AAV2/9-KAP1-GFP) and its mutations (AAV2/9-KAP1-S824A-GFP, AAV2/9-KAP1-S824D-GFP) were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Viral-mediated KAP1 and its mutations (injected at a titer of 109 VG/µl) were administered to the rats intracerebroventricularly (i.c.v) (bregma, 1.5 mm lateral, 0.8 mm posterior, and 3.8 mm deep) 14 days before ischemia.KAP1 plasmids (WT, S824D, S824A) were obtained from Shanghai Sangon, and were transiently transfected into cells with Lipo3000 liposome transfection reagent following the manufacture’s protocols. Briefly, 60% - 80% confluent cells were prepared for transfection. Abandon the medium and wash with PBS for 3 times. Then, add Opti-MEM into every well. Next, prepare lipofectamine 3000 reagent and plasmid mixture. The corresponding plasmid DNA (including P3000) and liposome were diluted with Opti-MEM medium respectively according to the size of the perforated plate, and then the two were mixed after standing at room temperature for 5 min. After that, incubate at room temperature for 15 min. Finally, the plasmid DNA-liposome complex was added into the cells in each well and cultured in the cell incubator for 24 - 48 h. The transfection efficiency was confirmed by Western blot analysis.
Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14000 × g at 4°C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Total proteins (50 µg) were separated by SDS-PAGE and electro-transferred to polyvinylidenedifluoride membranes (PVDF; Millipore, Billerica, MA, USA). Then the membranes were blocked with TBST buffer (NaCl 150 mmol/L, Tris 10 mmol/L, Tween-20 0.05% (v/v) pH = 7.6) containing 5% non-fat milk powder at room temperature for 2 h. The membranes were then probed overnight at 4°C with primary antibodies including phospho-KAP1(1:1000, Abcam, ab70369), KAP1(1:1000, Cell Signaling Technology, #4123), PCNA (1:2000, Servicebio, GB11010), Bcl-2 (1:1000, ABclonal, A0208), Bax(1:1000, ABclonal, A0207) followed by incubation with corresponding HRP-conjugated secondary antibodies (1:5000, Proteintech, SA00001-2) at room temperature for 2 h. Proteins on the membranes were visualized by adding the enhanced chemiluminescence reagent (Millipore, USA). Band intensities were analyzed using Image J 1.25 software (National Institutes of Health, Bethesda, USA) and normalized to that of loading controls.
Immunoprecipitation experiments were carried out with protein lysates using Beyotime Western and IP cell lysis buffer containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14000 × g at 4°C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Add a certain amount of the antibody into the protein lysates and shake at 4 ℃ overnight. The next day, wash the protein A/G magnetic beads (MCE) with 0.5% PBST (1 × PBS + 0.5% Triton X-100) for three times, and then add it into the protein antigen-antibody mixture. Then, continue to shake at 4 ℃ for 2.5 h. After that, wash the protein A/G magnetic beads-protein antigen-antibody complex with 0.5% PBST for five times. Finally, add 20-30 µl 2 × protein loading buffer into the complex, and then heat it in 100 ℃ water for 5 min. Samples were centrifuged at 12000 g for 2 min, and the supernatant was collected. Primary antibodies used in this stage including Flag (1:100, Proteintech, 20543-1-AP), KAP1 (1:100, CST, #4123), PCNA (1:100, Abcam, ab29), PCNA (1:2000, GB11010, Servicebio), Flag (1:10000, D110005, Sangon Biotech), CUL4A (Proteintech, 66038-1-Ig), α-Tubulin (1:1000, AF0001, Beyotime Biotechnology).
CCK-8 cell proliferation assay
The cells transfected with mutant plasmids were grown in 96-well plate. After attachment, cells were subjected to OGD/R or not. CCK-8 reagent and DMEM medium were mixed in 1:10. 100 µl of the mixture was added to each well, and then incubated in 5% CO2 incubator at 37 ℃ for 0.5 - 1 h. Then, the absorbance was determined at 450 nm with a microplate analyzer.
Bromodeoxyuridine (Brdu) staining
The cells transfected with mutant plasmids were grown in 24-well plate. After attachment, cells were subjected to OGD/R or not and were treated with or without Brdu for 8 h before staining. Brdu staining steps were as follows. Firstly, cells were washed with 0.3% TBS for 5 min × 3 times. Then, fix with 4% paraformaldehyde for about 20 min at room temperature. Next, cells were washed with TBS for 5 min × 3 times. Subsequently, 2 N hydrochloric acid was added to cells denaturing at room temperature for 1 h. After that, wash with TBS for 5 min × 3 times, and seal 1 h at room temperature (sealing fluid is 10% goat serum + 0.3% TritonX-100 PBS). Finally, incubate cells with the anti-Brdu antibody (1:500, Abcam, ab152095) at 4 ℃ overnight (diluent with 1% goat serum + PBS + 0.3% TritonX-100). The next day, wash with TBS for 5 min × 3 times and incubate with secondary antibody (1:1000, Invitrogen, A-21207) at room temperature for 2 h. Then, wash with TBS for 5 min × 3 times and stain with DAPI dye for 10 min. Finally, wash with TBS for 5 min × 3 times and photograph under Olympus IX73 inverted fluorescence microscope.
Annexin V-APC/7-AAD apoptosis assay
The cells transfected with mutant plasmids were grown in middle-plates, followed by OGD/R treatment or not after adherence. Digest and collect with trypsin free of EDTA, and wash with PBS twice to collect 1 - 5 × 105 cells. Cells were suspended with 500 µl binding buffer. 5 µl Annexin V-APC and 5 µl 7-Aminoactinomycin D (7-AAD) dye were added into cells. Incubate at room temperature in dark for 5 - 15 min. Then, samples were detected with Flow cytometry within 1 h.
Establishment of rat middle cerebral artery occlusion (MCAO) model
Male Sprague-Dawley rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged between 7 and 8 weeks old (180 g - 200 g) were used in this study. Rats were housed in the vivarium managed by the Division of Animal Lab Center of Xuzhou Medical University with a 12 h light/dark cycle and ad libitum access to food and water.
The MCAO model refers to the established method (Gong et al. 2015) in the laboratory, and the specific steps are as follows. After isoflurane inhalation anesthesia, the rat’s neck was vertically cut with scissors. Separate the surrounding tissue and expose rats left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Then, fasten CCA and ECA near the heart with sutures, clip ICA with arteriole clip. Subsequently, a small incision was made with ophthalmic scissors at an oblique 45° between the CCA ligation point and the three fork-lets at the upper end. The standard cord plug corresponding to body weight was carefully inserted at the incision point. The micro-arteriole was loosened, and the direction of the inlet line was adjusted to avoid the pterygopalatine artery, and the ICA was entered until the beginning of the middle cerebral artery (MCA). After ischemia for 1.5 h, the rats were under reperfusion for 24 h by pulling out the plug. The insertion depth of the thread plug in the sham operation group was about 5 mm, and the rest of the treatment was the same as that in the model group.
Balance beam test
The balance beam test was performed to assess the ability of rats to maintain balance and motor function while walking along an elevated and narrow strip of wood (122 × 2.5 × 42 cm). The performance was scored from 0 to 6 (no attempt to stay on the beam as 0 point); attempted to stay on the beam but no movement as 1 point; attempted to cross the beam but failed as 2 points; crossed the beam with contralateral hindlimb slips > 50% of the time as 3 points; crossed the beam with contralateral hindlimb slips < 50% of the time as 5 points; crossed the beam without slips as 6 points (Splenectomy fails to provide long-term protection against ischemic stroke. Aging Dis. 2018).
Open Field Test
A square cage (1 m × 1 m, with walls 45 cm high) containing an opaque acrylic container and a video camera were applied in the test. A rat was placed in the center of the square cage. The locomotive activities, including travel distance, speed, and time in corner were recorded (Breviscapine Alleviates Cognitive Impairments Induced by Transient Cerebral Ischemia/Reperfusion through Its Anti-Inflammatory and Anti-Oxidant Properties in a Rat Model). The whole arena was wiped clean with 70% ethyl alcohol between each test.
Frozen sections of brain tissue were performed using a Leica freeze slicer. Firstly, the brain was removed from the sucrose solution, and the bottom of brain was cut flat with a knife. Then, the back of the brain was placed facing the notch on the freezing platform and the abdomen facing operator vertically. Wrap the head from top to bottom with the embedding agent and freeze it in the machine for at least 2 h. After that, put the head on the freezer and cut into 20 µm thick slices. The brain slices were then placed in phosphate buffer saline (PBS; NaCl 137 mmol/L, KCl 2.7 mmol/L, Na2HPO4·12H2O 8 mmol/L, NaH2PO4·2H2O 1.5 mmol/L). If stored for a long time, the slices needed to be placed in PBS solution containing 60% glycerin and stored in a refrigerator at – 80 ℃.
Immunofluorescence staining of frozen sections
The rats were sacrificed 7 days after the MCAO model and the brain tissue was taken for follow-up study. The rat brain slices attached to viscous slide were washed with TBS (0.3% Triton X-100 PBS) for 5 min × 2 times, and sealed with blocking solution (0.3% Triton X-100 + 10% PBS of goat serum) at room temperature for 1 h. The rat brain frozen sections were then added with primary antibody working solution (Beyotime, P0023A) and incubated at 4 ℃ in a wet box overnight. The following day, wash the brain slides with TBS for 5 min × 5 times and incubated with secondary antibody solution (Beyotime, P0023D) in a wet box at room temperature for 1 - 2 h. Afterwards, wash the brain slides with TBS for 5 min × 5 times, and then DAPI staining was added to the brain slides incubating for 10 min. Finally, wash the brain slides with TBS for 5 min × 5 times, seal the slide with a sealing tablet and photograph under Olympus IX73 inverted fluorescence microscope.
The statistical analysis of data was conducted using GraphPad Prism 7. Experimental data were expressed as Means ± SEM. Students’ T test and One-way ANOVA was used for statistical analysis. Dunnet T test was used for comparison between multiple experimental groups and a control group. The data were checked for normally distributed by Kolmogorov-Smirnov test (with Dallal-Wikinson-Lillie for P value), before using parametric tests. P < 0.05 indicated that the difference was statistically significant.