Ca9 22 cells

Detailed information on the cell culturing procedure is provided previously [29 (link), 30 ]. We obtained normal human gingival fibroblasts (HGF-1) from the American Type Culture Company (ATCC number CRL-2014) and Ca9-22 cells (a cell line of oral epidermal gingival squamous carcinoma) from the Japanese Collection of Research Bioresources Cell Bank (JCRB number JCRB0625). The arecoline-conditioned medium (0.05–0.8 mM) was freshly prepared from arecoline hydrobromide (Sigma) in a growth medium (DMEM/F12). Cells were seeded into 96-well plates at a density of 10⁴ cells per well for 1 day and were then treated with various concentrations (0, 0.05, 0.1, 0.2, 0.4, and 0.8 mM) of arecoline for 24 h in a CO₂ incubator. MTT (Sigma) solution (5 mg/mL) was added to the cells and incubated for 2 h at 37°C. After the culture medium was removed, the cells were dissolved using DMSO, and absorbance was detected at 570 nm in an ELISA reader (Bio Tek el800), with a reference wavelength of 630 nm. The data are presented as the percentage of viable cells compared with the controls.

Ca9.22 cells derived from human gingival squamous cell carcinoma were purchased from the Japanese Collection of Research Bioresources Cell Bank (Shinjuku, Japan), and the cells were grown in DMEM/F12 (3:1 ratio) medium supplemented with 10% FBS, 1 × 10^–10 M cholera toxin, 0.4 mg/ml hydrocortisone, 5 μg/ml insulin, 5 μg/ml apo-transferrin, and 2 × 10^–11 M T3 in a humidified atmosphere of 5% CO₂ at 37°C. hFOB1.19 human fetal osteoblastic cells were cultured in DMEM/F12 without phenol red with 10% FBS, 1% antibiotic-antimycotic mixture and 0.3 mg/ml G418 at 34°C under humidified atmosphere of 5% CO₂.

P. gulae strains ATCC 51700 (fimA type A), D040 (fimA type B), and D049 (fimA type C) were selected from the stock culture collection in our laboratory^{7 (link),8 (link),24 (link)}. Bacterial cells were grown anaerobically at 37 °C for 24 h in trypticase soy broth supplemented with yeast extract (1 mg/ml), haemin (5 μg/ml), and menadione (1 μg/ml), as previously described^{48 (link)}; they were then used in the following experiments. Ca9-22 cells (originally isolated from human gingival epithelia) were obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan); these cells were used as an in vitro counterpart of gingival epithelial cells^{28 (link)} because they have been widely used as an in vitro culture model of gingival epithelial cells^{28 (link),49 (link)}. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum at 37 °C in 5% CO₂.