2.1 Culture of primary neural stem cells (NSCs) and neural progenitors (NPs)
Female C57BL/6 mice (25 g-30 g, 2-month-old) were purchased from SPF (Beijing) Biotechnology Co.,Ltd. and approved by the Institutional Animal Care and Use Committee of Capital Medical University. Cortex from embryonic day 14 (E14) mice were isolated and minced in a 0.25% trypsin solution for 5 min at 37 °C. The tissue was washed in media containing 10% FBS, and then passed through a 70 µm cell strainer. The extracted cells were resuspended in complete medium containing DMEM/F12, neurobasal, 2% B27, 10 ng/mL EGF, 10 ng/mL bFGF, 1% GlutaMAX and 1% penicillin/streptomycin, plated in Ultra Low Attachment six well Plates, and maintained at 37 °C in a humidified atmosphere containing 5% CO2. NSCs and NPs were subcultured by accutase every 2–3 days. The third passage cells were plated onto PDL–coated cell culture plates with complete medium.
2.2 Oxygen and glucose deprivation (OGD) operation
Cell medium was replaced by glucose-free Dulbecco's modified Eagles medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) in a a three-gas incubator containing 95% nitrogen and 5% CO2 at 37 °C for 1, 2, 4, 6, 8 h, respectively. Then cells were switched to glucose-containing medium and maintained at 37 °C, 5% CO2 incubator for 1, 2, 4, 7 days, respectively.
2.3 Cell viability assay
Cell viability was assessed by the CellTiter-Glo®Luminescent Cell Viability Assay. Briefly, equilibrate the plate and its contents at room temperature for approximately 30 minutes, then add a volume of CellTiter-Glo®Reagent equal to the volume of cell culture medium present in each well and mix contents for 2 minutes on an orbital shaker to induce cell lysis. Allow the plate to incubate at room temperature for 10 minutes to stabilize luminescent signal, and record luminescence with the luminometer.
2.4 EdU incorporation assay
Proliferation of NSCs and NPs was assessed by Click-iT® EdU Imaging Kits according to the manufacturer’s instructions. Isolated NSCs and NPs were seeded on 48-well-plates (4 × 105 cells/well) and cultured in medium without EGF and bFGF. After OGD operation, EdU was added to the medium and cells were cultured for another 4 days. Cultured cells were fixed with 4% PFA for 15 min, followed by permeabilization step using 0.5% Triton X-100. After permeabilization, the cells were incubated with Click-iT® reaction cocktails for 30 min in dark room. The number of total cells and Edu-positive cells were mounted using high content analysis.
2.5 Flow cytometry for assessment of EGFR and DCX
Neurospheres were dissociated by Accutase (Sigma). Select a fraction of cells from each tube to prepare a negative control tube (unmarked cells). Cells were fixed with 4% PFA for 30 minutes followed by wash with 1 ml PBS 0.15% BSA and incubation with 0.5% Triton X-100 for 30 minutes. Cells were washed once with 1 ml PBS 0.15% BSA and incubated with EGFR Antibody (Alexa Fluor® 750, Novus) or Doublecortin antibody (PE, biorbyt) for 30 minutes in dark. Cells were washed once and resuspended in PBS. Cells were then analysised on a BD FACSCalibur.
2.6 Real-time reverse transcription PCR (RT-PCR)
Purified RNA from NSCs and NPs were used as a template to synthesize cDNA using oligo-d (T) primers and SuperScript III /RNaseOUT Enzyme Mix (Invitrogen, Carlsbad, CA, USA). Relative gene expression was calculated via the 2 − ΔΔCT method, normalized, and expressed as fold change relative to U6 or β-actin. RT-PCR was performed in triplicate. Primers for myelin basic protein (Mbp): F: 5'-TCCGACGAGCTTCAGACCA-3', R: 5'-ACCCCTGTCACCGCTAAAGA-3'; primers for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase): F:5'-GCCTTCAAGAAAGAGCTTCG-3'; R: 5'-CAGATCACTGGGCCACAACT-3'; primers for microtubule associated protein-2 (Map-2): F: 5'-TCTCTAAAGAACATCCGTCAC-3', R: 5'-ATCTTCACATTACCACC TCC-3'; primers for β-tubulin-III (Tubb3) F:5' GCGCCTTTGGACACCTATT-3', R:5’-CCAGCACCACTCTGACCAA-3'; primers for glial fibrillary acidic protein (Gfap): F: 5'-AACAACCTGGCTGCGTAT-3', R: 5'-CTGCCTCGTATTGAGTGC-3'; primers for S100 calcium binding protein B (S100b): F:5'-CCCTCATTGATGTCTTCCACC3’, R:5'-TTCCTGCTCCTTGATTTCCTC-3'; primers for Epor F:5'-TCCTGGAGCACCTAT GACC-3', R:5'-CGAGATGAGGACCAGAATGA-3'; primers for βcr (Csf2rb) F:5'-TGGAGCAAGTGGAGCGAA-3', R:5'-CACAGCCAAAGCGAAGGAT-3'.
2.7 Co-Immunoprecipitation (Co-IP)
Co-IP assays were performed to identify the proteins. Briefly, 1 × 106 cells were collected and lysed in 300µL buffer containing nondenaturing lysis buffer, protease inhibitor and phosphatase inhibitors. For Co-IP using antibodies, before being added to the cell lysates, the antibodies were incubated with Protein A/G Magnetic Beads (Sigma-Aldrich) and IgG for 3 h at 4 °C to eliminate nonspecific binding. Then, the crosslinked Protein A/G Magnetic Beads were added to the cell lysates directly and incubated overnight at 4 °C. The magnetic beads were washed with IP wash buffer and collected. The protein complexes were eluted from the beads by 50 mM glycine (pH 2.8) and analyzed by Western blot.
2.7 Liquid chromatography tandem mass spectrometry (LC-MS/MS)
Protein bands were excised from the SDS-PAGE gels and were dissolved with 50 µl mobile phase A (H2O, 0.1% formic acid) and loaded onto a Acclaim PePmap C18-reversed phase column (75µm × 2 cm, 3 µm, 100 Ǻthermo scientific) and separated with reversed phase C18 column (75µm × 10 cm, 5 µm, 300 Ǻ Agela Technologies) mounted on a Dionex ultimate 3000 nano LC system. Peptides were eluted using a gradient : Start from 5% Buffer B ; 0 ~ 6 min 5%~8% Buffer B; 6 ~ 40 min 8–30% Buffer B; 40 ~ 45 min 30–60% Buffer B, 45–48 min 60–80% Buffer B; 48–56 min 80% Buffer B; 56–58 min increasing to 5% Buffer B; 58–65 min 5% Buffer B.at a flow rate of 400 nL min-1 combined with a Q Exactive mass spectrometer Thermo Fisher Scientific, Waltham, MA, USA). The eluates were directly entered Q–Exactive MS (Thermo Fisher Scientific), setting in positive ion mode and data-dependent manner with full MS scan from 350–2000 m/z, full scan resolution at 70,000, MS/MS scan resoltion at 17,500. MS/MS scan with minimum signal threshold 1E + 5, isolation width at 2 Da. To evaluate the performance of this mass spectrometry on the samples, two MS/MS acquisition modes, higher collision energy dissociation (HCD) was employed. And to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined 28%.
2.8 Western blot
Cell samples were processed for Western blot analysis as previously described. The PVDF membranes (Millipore corporation, MA, USA) were incubated with following primary antibodies at 4 °C overnight: anti-EPOR (rabbit, orb164257, Biorbyt, 1:1000), anti-βCR (mouse, 393281, Santa, 1:1000), anti-H3K9me3 (mouse, 5327, CST, 1:100). Antigen-antibody complexes were observed by enhanced chemiluminescence using an Immobilon Western blotting kit. The intensity of the bands was detected using a FluorChem®HD2 Gel Imaging System (Protein Simple, USA). The gray value of bands was analyzed by AlphaEase FC software (Alpha Innotech, CA, USA).
2.9 βCR siRNA transfection
NSCs and NPs were transfected with βCR siRNA using Lipofectamine Stem Transfection Reagent (Invitrogen, Carlsbad, CA, USA) for 48 h according to the manufacturer’s instructions and the transfection efficiency was validated by western blot.
2.10 Immunofluorescence analysis
Cells were fixed using 4% PFA for 15 min. After being washed three times in PBS, they were incubated with 3% BSA for 1 h. Cells were immunostained with primary antibodies of anti-EPOR (rabbit, orb164257, Biorbyt, 1:100), anti-βCR (mouse, 393281, Santa, 1:100), anti-H3K9 (mouse, 5327, CST, 1: 100) and anti-Syne-1 (rabbit, ab192234, abcam, 1:100). After incubating with secondary antibodies conjugated with DyLight 488 or Cy3 (Jackson ImmunoResearch), cells were counterstained with DAPI. The images were digitized using an Olympus Fluoview FV1000 microscope (Olympus, Japan).
2.11 Statistical analysis
Statistical analysis was performed using SPSS 23.0 software. Numeric data are presented as the mean standard deviation. Student’s t test was used for two-group comparisons. One-way ANOVA with Tukey–Kramer post hoc test was used for comparisons among several quantitative variables. The correlation between two variables was assessed using Pearson’s correlation test. Data for neurobehavioral tests were analyzed by a two-way repeated measures (RM) ANOVA followed by the Bonferroni post hoc correction. The correlation between two variables was performed using the Pearson’s correlation test. P < 0.05 was considered statistically significant.