2.4. Cell Cultures and Cytotoxicity Assay

In this study, two human oral cancer cell lines (OECM-1 and HSC-3), human dysplastic oral keratinocyte (DOK) representing OPMD cells and normal human oral keratinocytes (HOK), were incubated at 37 °C in a 5% CO₂ incubator. The human oral squamous carcinoma cell line OECM-1 (from a Taiwanese betel quid chewer) and HSC-3 tongue squamous carcinoma cell line are well-established models for squamous cell carcinoma [20,21]. OECM-1 and HSC-3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM). DOK cells were incubated in high glucose DMEM medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM glutamine (HyClone, Logan, UT, USA), 5 µg/mL hydrocortisone (HyClone, Logan, UT, USA), 100 µg/mL streptomycin (HyClone, Logan, UT, USA), and 100 U/mL penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA). Normal HOK cells (ScienCell, Carlsbad, USA) were incubated in oral keratinocyte medium (OKM, Cat. #2611, ScienCell, Carlsbad, USA). The medium of cell culture was refreshed every three days. Detailed information on cell culture has been reported in our previous study [20,21].

We added different concentrations (0, 20, 40, 60, 80, and 100 µM) of arecoline to HOK for 24 h to assess cell viability (Figure S1). We added MTT solution (5 mg/mL) to the cells and incubated them for 2 h at 37 °C in a CO₂ incubator. Subsequently, the viable cell percentage compared with vehicle controls was calculated by using an ELISA reader (el800, Bio Tek, Winooski, VT, USA).