Pcs 200 014

Human OSCC cell lines derived from oral cancer (SAS, HSC-2, HSC-3, Ca9-22, OSC-19, OSC-20, SAT, and KON) were purchased from the National Institute of Biomedical Innovation (Osaka, Japan). The HOC-313⁵⁴ (link) and TSU⁵⁵ (link) cell lines were kindly provided by Professor Kawashiri (Kanazawa University). HNOK cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA; PCS-200-014). SAS-R and HSC-2-R, which were established from SAS and HSC-2 cells, were used as the CRR cell lines. The CRR cell lines were produced by exposing cells to gradually increasing X-ray doses.⁵ (link) The OSCC cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; D6429; Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37°C and 5% CO₂. HNOK cells were cultured in dermal cell basal medium (PCS-200-030; ATCC) supplemented with the Keratinocyte Growth Kit (PCS-200-040; ATCC). CRR cells continued to proliferate under a daily IR dose of 2 Gy for >30 days in vitro.

MDA-MB-231 and BT-20 cell lines were obtained from late Dr. Gary Kruh (University of Chicago, Illinois). Human primary epithelial gingival keratinocytes (HPK), and normal colon fibroblast CRL1459 cells were purchased from American Type Culture Collection (ATCC#PCS-200-014, and ATCC#CCD-18Co—CRL-1459, respectively, Manassas, VA, USA). These cells were grown as an adherent monolayer with Dulbecco’s Modified Eagle’s Medium (Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (Biofluids Technologies, Port Richey, FL, USA), 2 mM of L-glutamine and 100 U/mL of penicillin and 100 µg/mL of streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described [42 (link)]. HPKs cells were cultured in dermal cell basal medium (ATCC# PCS-200-030) supplemented with factors from Keratinocyte growth kit (ATCC# PCS-200-040) as per manufacture’s protocol. These cells were cultured in a humidified incubator containing 5% CO₂ at 37 °C. All cells were assessed and confirmed to be free of fungi and mycoplasma. Cells were obtained from frozen stocks and cell passaging (up to P4) was performed at 80% cell confluency trypsin + 2.2 mM EDTA in phosphate-buffered saline (PBS).

HGECs were cultured according to the manufacturer’s protocol (PCS-200-014, American Type Culture Collection (ATCC), Manassas, VA, USA). The cells were sub-cultured in Dermal Cell Basal Medium (PCS-200-030, ATCC, Manassas, VA, USA) supplemented with Keratinocyte Growth Kit (PCS-200-040, ATCC, Manassas, VA, USA) and 100 U penicillin/streptomycin (P4333-100ML, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in humidified air with 5% CO₂. A confluent culture of HGECs was stimulated with P. gingivalis (10% formalin-fixed, strain 3327, ATCC, Manassas, VA, USA) (107 colony-forming units/mL) for 12 h to examine mRNA expression. As a negative control, HGECs were cultured without bacterial stimulation. P. gingivalis treatment was performed as previously described [20 (link)].
Bone marrow mononuclear cells (BMMCs) were collected from the femurs and tibias of C57BL/6J Jcl mice by density gradient centrifugation with Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA) in complete alpha-modified Eagle’s minimum essential medium (Sigma-Aldrich), containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (penicillin, streptomycin, and gentamicin; Invitrogen, Carlsbad, CA, USA).