Apigenin

Cells were seeded into 96-well plate at a density of 1 × 10⁴ cells per 100 μL culture medium for 24 h. After cell attachment, culture medium with apigenin at varying concentrations (0, 1, 10, 25, 50, 100 μmol/L) in dimethyl sulphoxide (D26650, DMSO, Sigma-Aldrich) was used to treat the cells for 24 h. apigenin (≥99.22% purity by LCMS, Figure S1) was procured from MedChem Express (#HY-N1201, Monmouth, NJ, USA). At the end of incubation, the medium was discarded, and the cells were washed with Dulbecco’s phosphate-buffered saline (#14190-144, DPBS, pH 7.4, Thermo Fisher Scientific Inc.) twice. To each well, 90 μL fresh culture medium and 10 μL presto/blue (A13262, Thermo Fisher Scientific Inc.) were added. After 4 h of incubation at 37 °C, the absorbance of the samples in the plates was measured at 570 nm with a reference wavelength set at 600 nm for PrestoBlue, using a microplate reader (Thermo Fisher Scientific, MA, USA). Cells treated with the vehicle control (0.1% DMSO in the culture medium) were regarded as 100% viable, and the viability of the apigenin-treated cells was determined.

The standard compounds including Ra, Lu, gallic acid, caffeic acid, and apigenin were obtained from MedChemExpress company (Monmouth Junction, NJ, USA). A recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (ab273068) was purchased from Abcam company (Cambridge, UK). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco company (Grand Island, NY, USA). The fetal bovine serum was purchased from Thermo Scientific company (Waltham, MA USA). The MTT or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye and mouse anti-beta-actin primary antibody were purchased from Sigma-Aldrich company (St. Louis, MO, USA). The TRI reagent^® was purchased from Merck Millipore company (Billerica, MA, USA.). The ReverTra Ace^® qPCR Master Mix was purchased from Toyobo Co., Ltd. (Osaka, Japan). The SensiFAST SYBR Lo-ROX Kit was purchased from Meridian Bioscience^® company (Cincinnati, OH, USA). rabbit anti-NLRP3 primary antibody, anti-ASC primary antibody, anti-caspase-1 (p50 and p20) primary antibody, anti-p-JAK1 primary antibody, and anti-p-STAT3 primary antibody and goat horseradish peroxidase-conjugated anti-mouse- or anti-rabbit-IgG were obtained from Cell Signaling Technology company (Danvers, MA, USA).

The human OSCC cell lines UM-SCC6 and Cal-27 were cultured in DMEM containing 10% fetal bovine serum (FBS; Gibco-BRL). UM-SCC6 cells were derived from human HNC patients and were obtained from Dr. Thomas Carey at the University of Michigan. Cal-27 cells (cat.no. CC0701) were purchased from Cellcook Company (Guangzhou, China). The following antibodies and reagents were used in the study: anti-GAPDH antibody (Santa Cruz Biotechnology, Cat. no. SC-25778 1:2000), anti-Mcl1 antibody (Cell Signaling Technology, Cat. no. 94296, 1:1000), anti-Ub antibody (Santa Cruz Biotechnology, Cat. no. SC-47721, 1:1000), anti-KLF10 antibody (GeneTex, Cat. no. GTX108661, 1:200), apigenin (Cat. no. HY-N1201, MedChemExpress).