All C57BL/6 mice (9–10 weeks, 19–25g) procedures were approved from the Animal Laboratory Center of Shanxi Medical University, Taiyuan, People's Republic of China. The procedure was in accordance with the "Guidelines for the Use of Nursing Animals" issued by the National Institutes of Health (NIH, 8th Edition, 2011). The experimental protocol was approved by the Experimental Animal Management Committee of Shanxi Medical University. The mice were maintained on a 12-hour light/dark cycle (lights on from 8:00 am–8:00 pm). On day 7.5 of pregnancy (E7.5), ethionine (Sigma-Aldrich, USA) was intraperitoneally injected only once at a dose of 500 mg/kg to establish the NTDs embryo model. And SAM (MedChemExpress, USA ) was intraperitoneally injected only once at a dose of 30 mg/kg. The same dose was intraperitoneally injected to the pregnant mice for control group.
Cell culture and treatments
Immortalized hippocampal neuron cell (HT-22), maintained in in DMEM (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA). All cells were incubated in atmosphere with 5% CO2 at 37°C. The cells were treated with 20 mmol/L ethionine, 2mmol/l SAM for 48 hours. The siRNA-METTL3 plasmid was synthesized by Sangon Biotech (Shanghai, China), and transfected into the cells using Lipofectamine RNAiMax (Santa Cruz) according to the manufacturer’s instruction. The sequence of siMETTL3 was forward: 5’-GCUGCACUUCAGACGAAUUTT-3’, reversed: 5’-AAUUCGUCUGAAGUGCAGCTT-3’, and that of the negative control (siCon) was forward: 5’-UUCUCCGAACGUGUCACGUTT-3’, reversed: 5’-ACGUGACACGUUCGGAGAATT-3’.
Enzyme-Linked Immunosorbent Assay for SAM and SAH
The concentrations of SAM and SAH in embryonic tissue were determined by enzyme-linked immunosorbent assay (ELISA; Elabscience®, wuhan, China).
m6A RNA Methylation Quantification
The m6A RNA Methylation Quantification Kit (Abcam, Massachusetts, US) uses colorimetry to quantify the m6A modification in the total RNA of the sample. Total RNA was isolated from embryonic brain tissues and its concentration were detected., Binded negative control, positive control and sample RNA to assay wells with Binding Solution for 90 min at 37°C according to the instructions. After the capture antibody was incubated at room temperature for 60 min, the detection antibody and enhancement solution were added and incubated for 30 min respectively. The Developer Solution were added to each well and incubated for 10 min away from light at room temperature. When the color in positive control wells turned medium blue, added Stop Solution and detected the absorbance at 450 nm within 10 min.
TUNEL staining assay
Apoptosis was assessed using an In Situ Cell Death Detection kit, POD (Roche, USA). The E10.5 embryos paraffin sections was removed, incubated and cells were permeated at 37°C for 20 minutes. Then the TUNEL staining assay was performed as described previously (Zhang et al., 2020a).
The sections were identified by the standard immunofluorescence staining. The following primary antibodies were used: rabbit anti-Cyclin D1 (1:200; ab16663; Abcam, Massachusetts, US), rabbit anti-β-catenin (1:100, Cell Signaling Technology), PCNA (1:200, Santa Cruz Biotechnology, sc-56), TCF-4 (1:100, Santa Cruz Biotechnology, sc-166699), and secondary antibodies used as Goat Anti-rabbit IgG H&L (Invitrogen, A11011). Nuclei were counterstained with DAPI (Sigma-Aldrich). Images were captured with using a fluorescence microscope (Nikon, Tokyo, Japan).
Protein isolation and Western blot
Total protein was extracted from cells or embryo brain tissues by RIPA lysis buffer (Solarbio, Beijing, China) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, USA) and 10 mM PMSF (Solarbio, Beijing, China), and protein was assayed by the BCA protein assay kit (Thermo Scientific). 20 µg proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Which were blocked with 5% skim milk in PBST (PBS with 0.05% Tween-20) for 1 h at room temperature. The transferred proteins were reacted with primary antibody overnight at 4°C and then labeled with secondary antibody for 1 h at room temperature. The primary antibodies used were antibody against β-catenin (1:1000, Cell Signaling Technology), TCF-4 (1:100, Santa Cruz Biotechnology, sc-166699), phosphor-GSK-3β (Ser9) (1:1000, Cell Signaling Technology, D85E12), axin-2 (1:1000, Abcam, ab32197), C-myc (1:1000, Abcam, ab32072), Cyclin D1 (1:500, Abcam, ab16663), PCNA (1:200, Santa Cruz Biotechnology, sc-56), BCL-2 (1:1000, Abcam, ab182858), Cleaved Caspase-3 (Asp175) (1:1000, Cell Signaling Technology, 5A1E), rabbit anti-β-Tublin (1:1000; Abcam, Massachusetts, US) and secondary antibodies were goat anti–rabbit IgG (1:3000; ZB-2301; ZSGB-BIO, Beijing, China), and goat anti–mouse IgG (1:3000; ZB-2301; ZSGB-BIO, Beijing, China), and β-Tublin was used as a housekeeping control. The protein complexes were visualized using an enhanced chemiluminescent (ECL) blot detection system (ChemiDocTM Imaging Systems, BIO-RAD, USA) following the manufacture’s instruction.
Flow cytometric analysis of cell apoptosis and cell cycle
The Annecin V-FITC/PI apoptosis detection kit was used for the apoptosis assay (KeyGEN BioTECH, Nanjing, China). The cell cycle detection kit (KeyGEN, Suzhou, China) was used for the cell cycle analysis. Then the cell apoptosis and cell cycle assay were performed as described previously (Zhang et al., 2020b). Experiments were performed three times for each group. Both data analyze with FlowJo 7.6 software.
EDU analysis of cell proliferation
Cell proliferation was also estimated using Cell-Light Edu Apollo DNA in vitro Kit (RiboBio, Guangzhou, China). Proliferative cells were visualized and imaged using a Zeiss LSM 510 META Laser Scanning Confocal Microscopy (Nikon, Tokyo, Japan). Proliferative cells were counted in different optical fields (magnification ×200) selected in a random manner and analyzed by the software of Image J.
Bromodeoxyuridine analysis of cell proliferation
Cells were inoculated in a 24-well plate. Treated with SiMETTL3 and SiNC for 24 hours, washed 3 times with PBS for 5 minutes each time and placed them on a shaker with slight shaking. Added 1ml 4% polyoxymethylene at room temperature for 30min. Then added 2mol/L HCl in 37℃ for 10min, mixed in 1ml 0.1% TritonX-100 at room temperature for 5min; washed 3 times with PBS for 10min each time; used 10% goat serum to close at 37℃ for 1h. Added the diluted Bromodeoxyuridine antibody overnight at 4°C. The next day, after 2h incubation at room temperature, washed 3 times with PBS, added the corresponding secondary antibody at 37°C for 2h. Washed 3 times with PBS. Then added appropriate DAPI to the wells at room temperature for 8 min. Washed 3 times with PBS for 10min and observed under a microscope.
AO-EB analysis of cell apoptosis
Detection of apoptosis by AO-EB double staining (Solarbio, Beijing, China) was also performed. The cells were cultured in the 24-well plate; 24 h after with ethionine and SAM treatment, the residual medium and the non-adherent cells were removed by washing with PBS and adding fresh PBS to the cells. A volume (20 ml) of working solution per millilitre of PBS was added (according to the dosage, mixing AO solution and EB solution into the working volume at a 1:1 ratio). After incubation for 3 mins at room temperature, cells were observed using a fluorescence microscope (Nikon, Tokyo, Japan).
Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, CA) and the data was presented as mean ± SEM. Statistical differences were performed by Student’s t-test for two group comparisons and one-way ANOVA for more than two group comparisons. Moreover, in one-way ANOVA analyses, LSD t-test were used to estimate the significance of the results. Differences were considered statistically significant when the *p<0.05, **p<0.01 or ***p<0.001.