Baterial growth curve and concentration standard curve
S.anginosus strain ATCC33397(supported by the State Key Laboratory of Oral Disease, Sichuan University )was cultured aerobically at 37℃ in THB medium(Solarbio,China)and measure the absorbance every 2 hours. Then the relationship between concentration of bacteria solution and absorbance value was obtained by serial dilution plate counting method.
Cell culture
The oral tongue squamous cell carcinoma cells SCC15 (supported by Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences)were cultured in DMEM/F12 with 10% fetal bovine serum(Gibco, USA) containing 100U/ml penicillin and 100 μg/ml streptomycin at 37℃and 5% CO2.
Co-culture time and concentration
The S.anginosus supernatant in stable phases was used for the experiment ,treating with centrifugation at 4000rpm/min for 20 minutes and filtering twice with 0.22μm filters. Then, the treated supernatant was called filtrate S and the bacterial medium THB was treated in the same manner as a control, called filtrate T. The Subsequent experiments were carried out with filtrate S and filtrate T. A series of concentrations of filtrate S were applied to SCC15, then an optimal co-culture time and concentration were obtained by CCK-8 assay.
Experimental groups and manipulating approaches
Methods to set up SCC15/S.anginosus (SCC15/ S) experimental group, SCC15/THB (SCC15/T) conditional control group, SCC15/S+3-MA conditional control group, SCC15/3-MA negative control group and SCC15 blank control group. Meanwhile, SCC15 group did not do any treatment, while SCC15/S, SCC15/T and SCC15/3-MA acted with filtrate S, filtrate T and 3-Methyladenine (3-MA) for 16h respectively; SCC15/S+3-MA pretreated with 3-MA for 4 hours and then infected with filtrate S for 16h.
Autophagic response measurement
Monodansylcadaverine staining
Monodansylcadaverine(MDC) staining was an independent method to evaluate autophagy. SCC15 cells were seeded in confocal dishes. After each group was treated accordingly,10μLMDC stain was added and mixed, and incubated at 37℃ for 20 minutes away from light; Then washed with 300μL 1X wash buffer twice. Pictures were obtained with the confocal microscope (Thermo, USA).
Real time PCR analysis
Total RNA was isolated from SCC15 cells using Trizol method (Beyotime, China). cDNA was synthesized by qScript cDNA synthesis kit (Sigma, USA) and primers synthesized by Wuhan Sevier Biotechnology Co., Ltd. The sequences are shown in Table 1. Quantitative gene expression was performed for Beclin1, LC3 using one-step RT-PCR kit (Takara, Japan). Expression values were normalized to GAPDH.
Western blot analysis
The cell samples were collected and lysed in RIPA with PMSF (Beyotime, China). Then, protein concentration was mearsured by BCA protein kit (Beyotime, China). The cell lysates were separated by 10%SDS–PAGE and transferred to the 0.22um PVDF membranes. The bands were detected by enhanced chemiluminescence (ECL) after the primary antibody at 4℃ for a night and second antibody at room temperature for 1 hour. The antibodies used in this study include: anti-LC3, anti-Beclin1 (Cell Signaling, USA) and GAPDH (Beyotime, China).
Cell proliferation analysis
Cells were seeded in a 96-well plate at a density of 5×103/well and incubated at 37℃for 24h ,then each group was treated accordingly. After that,10μL CCK-8/well with 100 μL DMEM/F12 was added and incubated at 37℃ for 4h. Absorbance was determined at 450nm with a microplate reader.
Cell cycle and apoptosis analysis
The cells were digested with trypsin and washed with cold PBS, then fixed overnight in 70% ethanol at -4℃. Ethanol-fixed cells were collected and washed with PBS, then cell counting. 106cells of each group were centrifuged and 0.5 mL PI/RNase stain was added to cell pellets. After 30 minutes at room temperature in the dark, the cell cycle was analyzed with flow cytometre with a 488nm argon laser.
Cell culture medium of each group was collected respectively and Cells were trypsinized with EDTA-free trypsin. Then washed cells with cold PBS twice and cell counting. 106cells of each group were centrifuged and 400 μL 1x AnnexinV binding buffer was added to resuspend cells. Afterwards, added 5μL FITC and 10 μL PI per group for incubation at -4℃ in the dark for 15minutes and 5minutes respectively.
Cell migration and invasion assay
The cell migration was measured by wound healing assay. Scratched a line with pipette tip in the middle of each well and washed with PBS 3 times slightly. Then 2 mL of serum-free medium was added to each well and incubation at 37 ° C for 24 hours. The pictures of each group were recorded at 0h, 6h, and 12h under microscopy at 40 magnifications.
The cell invasion was measured by the transwell system (BD, USA). Treated cells were inoculated in the upper chamber with free-FBS medium, and F12 culture medium containing 10% FBS was added to the lower chamber. Additionally, an insert covered with Matrigel was used for invasion measurements. After 24 hours, cells migrated to the opposite side of the insert were stained with DAPI and quantified.
Statistical analysis
Data analysis was performed by SPSS 25.0 software. All values are calculated and expressed as mean ± standard deviation (SD). Anaylsis of Variance(ANOVA)and SNK-q test were used to compare between the groups. P-value<0.05 was considered statistically significant.