Cell lines and reagents
In this study, human OSCC cell lines including WSU-HN4, WSU-HN6 and CAL-27 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, New York, NY, USA). Zymosan was purchased from Sangon Biotech (Shanghai, China). Candida albicans ATCC 90028 was cultured on Sabouraud Dextrose Agar (SDA).
CCK-8 cell viability assay
Cell viability assay was performed with CCK-8 (Dojindo, Kumamoto, Japan). OSCC cells (5,000 cells/well) were seeded into 96-well plates and cultured for appropriate time before treatment while the controls were treated with 100μL PBS. After treating targeted intervals, 10μL CCK-8 reagent was added, and optical density was read at 450 nm on a microplate reader (Bio-Rad, Hercules, CA, USA) after incubation for 1.5 h.
Cell adhesion assay
OSCC cell suspension was seeded onto a coverslip placed in a 24-well plate, which was disinfected by UV light, and the cells were incubated for 24 hours to attach to coverslip, and then stimulated by zymosan or PBS for additional 24 hours. Wash the cells with PBS before adding C. albicans suspension in PBS, and incubate at 37oC with 5% CO2 for 1 hr. After incubation, the DMEM with unattached bacteria was aspirated, and each well was washed with PBS and then fixed with in 95% ethanol for one hour. After that, the coverslip was taken out for Gram staining.
Cytokine detection
The OSCC cells were seed into a 12-well plate before treatment with PBS or zymosan, and then supernatants were collected to measure the concentration of IL-1β, IL-6, IL-8 and TNF-α by IMMULITE®1000 (SIEMENS, German) according to the manufacturer's instructions.
Quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (Invitrogen, San Diego, CA, USA), and real-time PCR amplification was carried out by the two-step reaction. Firstly, cDNA was synthesized by GeneAmp® PCR system 9700 (Applied Biosystems) with PrimeScriptTM RT Reagent kit (TaKaRa, Shiga, Japan), and then real-time PCR was performed in the 7500 system (Applied Biosystems) with the SYBR Premix Ex Taq II (TaKaRa). The experiment was repeated in triplicate on independent occasions. The GAPDH gene was used for the normalization of gene expression, and relative expression of TLR2, MyD88, NLRP3, ASC, Caspase-1 and IL-1β was determined using the 2−ΔΔCt method.
Western bolt
The OSCC cells treated with zymosan (100μg/mL) were collected and lysed on ice in RIPA buffer with phosphatase inhibitor (Solarbio, Beijing, China) and phenylmethanesulfonylfluoride (PMSF). After centrifugation, the supernatants were determined by Enhanced BCA Protein Assay Kit (Beyotime, Haimen, China) and then were incubated at 100 °C for 10 min. Equal amounts of total protein were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% nonfat milk powder in Tris-buffered saline/Tween 20 (TBST) for 1 hour at room temperature and incubated in primary antibody overnight at 4℃, and then incubated with HRP-conjugated secondary antibodies (anti-rabbit IgG from Sigma-Aldrich and anti-mouse IgG from Cell Signaling Technology) at a dilution of 1:5,000. Protein bands were visualized using High-sig ECL substrate and Tanon 5200CE machine (Tanon, Shanghai, China).
Immunofluorescence assay
The OSCC cells treated with PBS and zymosan deposited on glass slides were fixed with 4% paraformaldehyde for 20min, and then permeabilized with 0.1% Triton X-100 for 3min and blocked with 5% bovine serum albumin for 30 min. The slides were incubated with anti-TLR2 antibody (1:10, abcam, MA, USA) or anti-NF-κB p65 antibody (1:400, Cell Signaling Technology, MA, USA) at 4 °C overnight. Anti-mouse IgG antibody with Alexa Fluor 568 and anti-rabbit IgG with Alexa Fluor 488 served as the secondary antibody (Thermo Fisher Scientific, USA). After washing three times with TBST, the cells were stained with DAPI for 3 min to visualize the nuclei. Photos were taken by an inverted microscope equipped with fluorescence optics (Olympus, Osaka, Japan).
Statistical analysis
The data were displayed as the mean ± standard deviation, and processed by GraphPad Prism 5 (Version 5.01, GraphPad Software, CA, USA). Statistical analysis was conducted by SAS 8.2 (SAS Institute Inc., Cary, NC, USA). It was considered statistically significant if the two-tailed P-value was less than 0.05 and indicated * when P<0.05, ** when P<0.01, *** when P<0.001 and **** when P<0.0001.