Hsc 2

The human OSCC cell lines HSC2, HSC3, HSC4, and KON were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan, and SCC25 cells were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. All cell lines were authenticated by JCRB and ATCC using short tandem repeat (STR) analysis. Total RNA from the normal tongue was purchased from Biochain (Newark, CA, USA) and used as a control. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan) in 5% CO₂ in air at 37°C. Anti-MIA antibody (R&D Systems) was used for neutralizing MIA in cultured medium at 2 μL/mL concentration. Further, 20 mM of recombinant human MIA (rhMIA) (Abnova, Taipei, Taiwan) treatment was performed. Moreover, cells were treated with 10 nM paclitaxel (Wako Pure Chemical), cisplatin (Wako Pure Chemical), and 5-FU (Wako Pure Chemical).

The human HNSCC cell lines HSC-2, HSC-3, and HSC-4 were obtained from the Japanese Collection of Research Bioresources cell bank (Osaka, Japan). SCC-9 and SCC-25 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The prostate cancer cell line DU145 was obtained from the NCI Division of Cancer Treatment and Diagnosis (Bethesda, MD, USA). HSC-2, HSC-3, and HSC-4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; #D5796, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; #FBS-12A, Capricorn Scientific, Ebsdorfergrund, Germany) at 37°C in a humidified atmosphere of 5% CO₂. SCC-9 and SCC-25 cells were cultured in DMEM/Nutrient Mixture F12 medium (containing L-glutamine and sodium bicarbonate without HEPES; the medium was liquid, sterile-filtered, and suitable for cell culture; DMEM/F12; #D8062, Sigma) supplemented with 10% FBS and 400 ng/mL hydrocortisone (#088-02483, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) at 37°C in a humidified atmosphere of 5% CO₂. DU145 cells were cultured in RPMI medium containing 10% FBS at 37°C in a humidified atmosphere of 5% CO₂.

Six OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22, Ho-1-N-1, and Ho-1-U1), which were used for this study, were provided by the Japanese Collection of Research Bioresources Cell Bank. Cells were grown in RPMI 1640 supplemented with 10% FBS at 37°C and 5% CO₂.

The human oral squamous cell carcinoma cell lines, HSC-2 (JCRB0622: oral squamous carcinoma cells derived from weak gingivae) and HSC-3 (JCRB0623: oral squamous carcinoma cells derived from highly advanced tongue cancer) (JCRB Cell Bank, Tokyo, Japan), human cervical adenocarcinoma cells (HeLa) (TKG0331, Cell Resource Center for Biomedical Research in Tohoku University, Miyagi, Japan), and the normal human keratinized epithelial cell line, HaCaT (Cosmo Bio Co., Ltd., Tokyo, Japan), were used in the present study. All cell lines were authenticated using a short-tandem repeat analysis. All cells were cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM) (041-29775, Fujifilm-Wako Pure Medical Corporation, Miyazaki, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, New York, NY, USA), 100 mg/mL streptomycin, and 100 U/mL penicillin (Fujifilm-Wako Pure Medical Corporation, Miyazaki, Japan) at 37 °C in a humidified atmosphere with 5% CO₂.

SAS, OSC-19, SCC-4, HSC-2, HSC-3, HSC-4, and KOSC-2 cl3-43 (KOSC2) human oral cancer cells and human embryonic kidney 293 (HEK 293) cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The Jurkat E6.1 (Jurkat) human T-cell lymphoma cell line was purchased from KAC Co., Ltd. (Kyoto, Japan). SAS, KOSC2, and Jurkat cells were grown in RPMI supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). Anti-GART rabbit monoclonal antibody (H8132) for western blot analysis and anti–β-actin mouse monoclonal antibody (ab6276) were purchased from Abcam (Cambridge, UK). Anti-GART mouse monoclonal antibody (4D6-1D5) (Abnova, Taipei, Taiwan) for immunofluorescence cytochemistry, anti-BTK mouse monoclonal antibody (ab54319), and anti-Bmx goat polyclonal (sc-8874) (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were purchased from the suppliers indicated. ITK inhibitor (Cmpd-5) was obtained from Carna Biosciences, Inc. (Hyogo, Japan) (Figure S1).

SAS, HSC3, SCC-4, OSC20, HSC4, OSC19, HSC2, KON, Ca9-22, SAT, and Ho-1-U-1 were obtained from JCRB (Japanese Collection of Research Bioresources Cell Bank). These cells were maintained in Dulbecco's Modified Eagle's Medium (FUJIFILM Wako) supplemented with heat-inactivated 10% fetal bovine serum (Nichirei Bioscience Inc., Tokyo, Japan) and 1% Penicillin and Streptomycin (FUJIFILM Wako) at 37 °C in 5% CO₂. For growth assay, 5 × 10³ cells were plated onto 96-well plates, and measured cell proliferation by using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), according to the manufacturer’s instructions.