Cell lines and reagents
In this study, the human OSCC cell lines WSU-HN4, WSU-HN6 and CAL27 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, New York, NY, USA) and 1% penicillin-streptomycin. All cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Zymosan was purchased from Sangon Biotech (Shanghai, China). C. albicans ATCC 90028 was cultured on Sabouraud Dextrose Agar (SDA) at 37 °C in an incubator containing 5% CO2.
CCK-8 cell viability assay
The 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 12-24 h before treatment. The OSCC cells were treated with zymosan (10 μg/ml and 100 μg/ml) or with 100μL PBS as the control. After 12 h, 24 h or 48 h of treatment, 10 μL of CCK-8 reagent was added, and the 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 h to attach to coverslip, and then stimulated by zymosan or PBS for an additional 24 h. The cells were washed with PBS before adding C. albicans suspension in PBS, and were further incubated at 37 oC with 5% CO2 for 1 h. After incubation, the DMEM with unattached yeast was aspirated, and each well was washed with PBS and then fixed with 95% ethanol for one h. After that, the coverslip was removed for Gram staining.
Cytokine detection
OSCC cells were seeded into a 12-well plate before treatment with PBS or zymosan, and then supernatants were collected to measure the concentrations of IL-1β, IL-6, IL-8 and TNF-α. These cytokines were measured by chemiluminescence immunoassay (CLIA) using IMMULITE®1000 (SIEMENS, Germany) with commercial reagents 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 a two-step reaction. First, 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 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 the relative expression of TLR2, MyD88, NLRP3, ASC, Caspase-1 and IL-1β was determined using the 2−ΔΔCt method.
Western blot
OSCC cells treated with zymosan (100 μg/mL) were collected and lysed on ice in RIPA buffer with phosphatase inhibitor (Solarbio, Beijing, China) and phenylmethanesulfonylfluoride (PMSF). After centrifugation, the protein concentration in supernatants was determined by an Enhanced BCA Protein Assay Kit (Beyotime, Haimen, China), and the protein samples were then 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 h at room temperature and incubated with primary antibody overnight at 4 ℃ (TLR2, ab16894, Abcam; MyD88, ab133739, Abcam; E-cadherin, ab40772, Abcam; NF-κB p65, #8242, Cell Signaling Technology; p-NF-κB, #3033, Cell Signaling Technology and β-actin, A1978, SIGMA) 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 for 1 h at room temperature. Protein bands were visualized using High-sig ECL substrate and a Tanon 5200CE machine (Tanon, Shanghai, China).
Immunofluorescence assay
The OSCC cells treated with PBS or zymosan and placed on glass slides were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 3 min and blocked with 5% bovine serum albumin for 30 min. The slides were incubated with anti-TLR2 antibody (1:10, Abcam, MA, USA) at 4 °C overnight. Anti-mouse IgG antibody with Alexa Fluor 568 served as 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. Images 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). Two-tailed P-value less than 0.05 was considered statistically significant and was indicated with * when P<0.05, ** when P<0.01, *** when P<0.001 and **** when P<0.0001.