Firstly, we confirm that all methods were performed in accordance with the relevant guidelines.
The existence of a vaginal plug of Sprague–Dawley (SD) rats was treated as E 0.5 d. One day after birth was recorded as PN1. All experiments were conducted in accordance with the scheme approved by the Ethics Committee of Southwest Medical University (The number accepted is 201,903–187, the original document is shown in related files), including any relevant details, and we confirmed that all experiments were performed in accordance with relevant guidelines and regulations.
HE and immunohistochemistry staining
The SD rat embryo heads or maxillas of E12.5 d, E15.5 d, E19.5 d, PN1, and PN4 (n = 3 each group) were collected and fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 24 h. The maxilla samples were demineralized with ethylenediaminetetraacetic acid (EDTA) decalcifying solution (Solarbio). Then, the samples were dehydrated by alcohol, embedded in paraffin and sliced into 5 μm tissue sections. HE staining was carried out according to the manufacturer’s protocols of HE Staining Kit (Solarbio). After dewaxing and rehydration by xylene and gradient alcohol, the slides were dyed with hematoxylin solution for 3 min, then rinsed with water for 3 min to remove float color. Then the slides were dyed with eosin solution for 30 s and rinsed with water for 3 s. Finally, dehydrated with gradient alcohol, transparented with xylene and sealed treatment. Scanning and analysis of staining results were performed with a slice digitizing scanner (OLYMPUS, Japan). For immunostaining, briefly, after dewaxing and rehydration, the slides were repaired with sodium citrate antigen repair solution (Bioss, Beijing, China), and then incubated with 3% H2O2 solution to eliminate endogenous peroxidase activity. The slides were blocked with goat serum (Bioss) and then incubated with the rabbit polyclonal antibody to Mage-D1 (1:100, Biorbyt, Cambridgeshire, UK) at 4 °C overnight. Next day, the slides were incubated with an anti-rabbit secondary antibody (1:3000, Bioss), followed by colouring with diaminobenzidine (DAB) (Zsbio, Beijing, China). Finally, cell nuclei were counterstained with haematoxylin (Solarbio). Following dehydrated, sealed and other steps are the same as HE staining.
Isolation and culture of E19.5 d EMSCs
The isolation and culture of E19.5 d EMSCs were carried out as previously described1,5. The embryonic maxillofacial process of E19.5 d SD embryo rats was dissected, and the maxillary dental germ was taken. The minced tissue was placed directly in a sterile petridish using the tissue block adherence method. Then, the complete culture medium (composed of 89% Dulbecco’s modified eagle medium/F12 (Sigma, Darmstadt, Germany), 10% foetal bovine serum (Ausgenex, Gold Coast, Australia) and 1% penicillin–streptomycin liquid (Solarbio)) was gently added and cultured in a humidified incubator at 37 °C and 5% CO2 for approximately 3 days. After the cells had fully crawled out, routine follow-up cell passaging treatments were carried out.
After the cells reached 70% confluence in a six-well plate, they were fixed with 4% paraformaldehyde for 30 min and washed gently with PBS buffer three times, then 0.1% Triton X-100 (Solarbio) was added to break the membrane for 20 min and washed again. Approximately 100 μm of phalloidin working solution (Sigma) was added, followed by incubation for 2 h at room temperature. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole dihydrochloride solution (DAPI) (Solarbio) and incubated for 10 min in the dark. Finally, the cytoskeleton was observed with a confocal laser scanning microscope (CLSM) (Leica CS SP8, Heidelberg, Germany).
Flow cytometry identification
Approximately 5 × 105 cells were collected in each group, and then we detected cell surface markers by flow cytometry. The cells were fixed with 4% polyoxymethylene for 15 min, and then primary antibodies (mouse monoclonal antibody to CD44, CD29, CD90, CD105, CD146 and p75NTR) (1:100; Santa Cruz Biotechnology, Texas, USA) were added and incubated overnight at 4 °C. The anti-mouse secondary antibody/FITC (1:100, Bioss) was added the next day and incubated for at least one hour. The cells were then analysed by CytoFLEX flow cytometry (Beckman Coulter, California, USA).
Transfection of Mage-D1-overexpressing and Mage-D1-silenced plasmids
The full-length coding region of rat Mage-D1 was amplified by PCR and cloned into the vector pLVX-puro for expression. The specific primers were as follows: 5′- ACACTCGAGATGGCTCAGAAACCGGACGGCG-3′ (forward) and 5′-CTGAAT TCTTACTCAACCCAGAAGAAGCCAATGGCACCG-3′ (reverse). The plasmids were cotransfected with psPAX2 and pCMV-VSV-G packaging plasmids into HEK-293 T cells with active growth by Lipofectamine 2000 (Invitrogen, Massachusetts, USA). The virus-containing supernatant was collected at 2 to 3 days after transfection, which was used to infect the target cells, the E19.5 EMSCs. Then the six-well plate cells reached 70% confluence, appropriate amount of virus solution and 8 μg/ml polybutene (Solarbio) was added to the cell medium. After 24 h, replace the culture medium containing virus solution with complete culture medium. Then the target cells were continuously screened for about 7 days with 4 μg/ml puromycin (Solarbio). The stable Mage-D1 overexpression of EMSCs was established for further research. To knock down the expression of endogenous Mage-D1, plasmids were established with pLKO.1. The sequences were as follows: 5′-AAGGTGGCCTTTAAGTCACAG-3′. The packaging of these knockdown lentiviruses is similar to that of overexpression lentiviruses.
The transfected cells were plated on a cell slide until the cells reached 70% confluence and then fixed with 4% paraformaldehyde, and then 0.1% Triton X-100 (Solarbio) was added to break the membrane for 20 min, followed by appropriate amount of 5% goat serum (Bioss) was added to each slide to sealed for 1 h at room temperature. After removing the sealing liquid, incubation with the rabbit polyclonal antibody to Mage-D1 (1:100, Biorbyt) overnight at 4 °C. The anti-rabbit secondary antibody/FITC (1:100, Bioss) was added the next day and incubated at room temperature for 30 min. For immunofluorescence double staining of paraffin sections, the steps of dewaxing to blocked were the same as those of immunohistochemistry. Then, two heterologous primary antibodies were diluted and mixed with PBS (the rabbit polyclonal antibody of Mage-D1,1:100, Biorbyt; the mouse monoclone antibody of 75NTR, 1:100, Santa Cruz; Dlx1, 1:100, Santa Cruz; Msx, 1:100, Santa Cruz), followed added to the tissue surface and incubated at 4 °C overnight. Next day, both the anti-rabbit secondary antibody/FITC (1:100, Bioss) of anti-mouse/Cy3 (1:100, Bioss) was added and incubated at room temperature for 1 h. The cell nuclei were counterstained with DAPI. Finlly sealed the cell slide with antifluorescent quencher and observed under CLSM (Leica).
CCK-8 proliferation and scratch test
Briefly, the transfected EMSCs were seeded in a 96-well plate at 2 × 103 cells/well (Corning). Starting on the second day, we detected cell proliferation by using the CCK-8 assay for 8 consecutive days according to the manufacturer’s instructions. The number of viable cells in each well was determined by measuring the absorbance at 450 nm wavelength with a microplate reader (Varioskan LUX Multifunctional Enzyme Marker, Thermo Fisher Scientific, California, USA). Cell proliferation was expressed as the mean ± standard deviation of the absorbance of 5 wells in each group. For scratch test, the transfected cells were spread in a six-well plate until full. Then, we changed to serum-free medium and used a 200 μL pipette tip to mark the inside of the well plate at least three traces and cultured the cells in a humidified incubator at 37 °C and 5% CO2. Photos were taken at 0 h, 24 h, and 48 h by inverted phase contrast microscope (Zeiss, Jena City, Germany). ImageJ software (1.8.0 for Microsoft, National Institutes of Health, Bethesda, USA) was used to analyse cell migration area ratio in each group.
Real-time PCR assay
General RNA of every group of cells was obtained by Trizol reagent (Invitrogen)55. After the cells were lysed by Trizol reagent, the supernatant containing RNA was separated by chloroform, and then isopropyl alcohol was added to precipitate RNA, which was washed with 70% ethanol, and finally RNA was dissolved in non-enzymatic water. RNA was reverse-transcribed into cDNA using the PrimeScript™RT reagent Kit with gDNA Eraser (TaKaRa, Kusatsu, Japan) according to the manufacturer’s instructions. The quantities of RNA and cDNA were detected with the molecular devices (Nanodrop 2000, Thermo Fisher Scientific). Quantitative real-time PCR was carried out by Fluorescence quantitative PCR instrument (Bio-Rad, California, USA) using SYBRII qPCR master mix reagent (TaKaRa) and running with a 20 µl reaction system. There were at least 3 secondary wells per group. At the same time, the GAPDH gene was used as a control. Primer information for related genes is shown in Supplementary Table 1.
Co-immunoprecipitation (Co-IP) and WB
Total cell proteins were retrieved with cell lysis buffer (Beyotime), and the protein level was measured by the BCA kit (Bioworld, Beijing, China). For coimmunoprecipitation, first, the magnetic beads (Bio-rad) were incubated with rabbit polyclonal antibody to Mage-D1 (1:200, Proteintech, Chicago, USA) for 2 h, followed by the addition of protein lysate and incubation overnight at 4 °C. The unreacted proteins were separated magnetically and discarded the next day. Western blot was conducted as described previously. The protein samples were separated by SDS–PAGE and transferred to PVDF membranes (Pierce, Dallas, USA). The blots on the membranes were cut prior to hybridization with antibodies during blotting. Primary antibodies against Mage-D1 (1:1000; Biorbyt), p75NTR (1:1000, Cell Signaling Technology, Poston, USA), Runx2 (1:1000, CST), BSP II (1:1000, CST), Dlx1 (1:200, Biorbyt), Msx1 (1:200, Biorbyt), and GAPDH (1:1000, CST) were used as internal standards. On the second day, the membranes were incubated with anti-rabbit or anti-mouse secondary antibody/HRP (1:5000, Bioss) for two hours at room temperature. Finally, the signal was revealed with BeyoECL Plus solution (Beyotime) by the imaging system (Bio-Rad). In order to see the difference clearly, the Quantity One software (Bio-Rad; Hercules, USA) was used to grayscale analysis for the blots and the ratio of the grayscale value of target blot and that of internal reference GAPDH blot was considered as the relative expression level.
Alkaline phosphatase and alizarin red staining
Each group cells were cultured with the osteogenic induction medium. On days 14 and 21, the cells were fixed with 4% paraformaldehyde and stained using an alkaline phosphatase (Alp) staining kit (Beyotime, Shanghai, China) or alizarin red (Sangon Biotech, Shanghai, China) staining kit separately, according to the manufacturer’s instructions and the previous study1. The stained images were captured by a stereo fluorescence microscope (Zeiss).
Total data are shown as the mean ± standard deviation (SD). Statistical significance was determined with SPSS 20.0 software (IBM Analytics, Armonk, USA) and GraphPad Prism8 software (GraphPad software, california, USA). The comparison between groups in our study were pairwise comparison. Following the determination of normal distribution by F-test, normally distributed data were analyzed by unpaired T test and non-normally distributed data were analyzed by Mann–Whitney test. Significance levels of p < 0.05 indicated significant differences.
The study is reported in accordance with ARRIVE guidelines.