Experimental animals. Sprague–Dawley (SD) rats were acquired from Chongqing Medical University Animal Laboratory in this study. The existence of a vaginal plug 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 the Affiliated Stomatological Hospital of Chongqing Medical University, 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 (Solarbio). For immunostaining, briefly, the slides were blocked with goat serum (Bioss, Beijing, China) and then incubated with the rabbit polyclonal antibody to Mage-D1 (1:100, Biorbyt, Cambridgeshire, British) 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). Scanning and analysis of staining results were performed with a scanner.
Isolation and culture of E19.5 d EMSCs. The isolation and culture of E19.5 d EMSCs were carried out as previously described.1,5 The embryonic maxillofacial process of E19.5 d SD embryo rats was dissected, and the maxillary tooth germ was taken. The minced tissue was placed directly in a sterile petridish using the tissue block adherence method. Then, the 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.
Phalloidin staining. After the cells reached 70% confluence in a six-well plate, they were fixed with 4% paraformaldehyde for 30 minutes, and then 0.1% Triton X-100 (Solarbio) was added to break the membrane for 20 minutes. Approximately 100 microlitres 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, 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 minutes, and then primary antibodies (mouse monoclonal antibody to CD44, CD29, CD90, CD105 and CD146) (1:100; Santa Cruz Biotechnology, Texas, USA) were added and incubated overnight at 4°C. The anti-mouse secondary antibody (1:100, Bioss) was added the next day and incubated for at least one hour. The cells were then analysed by flow cytometry.
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-293T 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, and 8 µg/ml polybutene (Solarbio) was added. After at least 24 hours, the target cells were continuously screened for 10 days with 4 µg/ml puromycin (Solarbio) 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.
Immunofluorescence staining. The transfected cells were plated on a cell slide until the cells reached 70% confluence and then fixed with 4% paraformaldehyde, followed by incubation with the rabbit polyclonal antibody to Mage-D1 (1:100, Biorbyt) overnight at 4°C. The anti-rabbit secondary antibody (1:100, Bioss) was added the next day and incubated at room temperature for 30 minutes. The cell nuclei were counterstained with DAPI and observed under CLSM.
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. Cell proliferation was expressed as the mean ± standard deviation of the absorbance of 5 wells in each group. 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 and cultured the cells in a humidified incubator at 37°C and 5% CO2. Photos were taken at 0, 24, and 48 h. ImageJ software 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) and then reverse transcribed with a kit (TaKaRa, Kusatsu, Japan). The quantities of RNA and cDNA were detected with an ultraviolet spectrophotometer. Quantitative real-time PCR was carried out by using SYBRII qPCR master mix reagent (TaKaRa) and run 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 (Biorad, California, USA) were incubated with rabbit polyclonal antibody to Mage-D1 (1:200, Proteintech, Chicago, USA) for 2 hours, 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). 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 (1:5000, Bioss) for two hours at room temperature. Finally, the signal was revealed with BeyoECL Plus solution (Beyotime).
Statistical analysis. Total data are shown as the mean ± standard deviation (SD). Statistical significance was determined with SPSS 20.0 software (IBM Analytics, Armonk, NY, USA). Statistical comparisons were made by Student's t test and one-way analysis of variance (ANOVA). Differences were statistically significant when p < 0.05.