α minimum essential medium

MGFs were isolated from healthy gingival tissue from the palate of BALB/c mice which were purchased from CLEA Japan, Inc. (Tokyo, Japan). The MGFs were cultured in Minimum Essential Medium α (Thermo Fisher Scientific, Wilmington, DE, USA) containing 10% fetal bovine serum (Hyclone Laboratories Inc, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 ℃ in a CO₂ incubator. When the cells reached confluency, they were separated by treatment with 0.25% trypsin-EDTA (Thermo Fisher Scientific) and collected by centrifugation. This study was approved by the Institutional Animal Care and Use Committees of Aichi Gakuin University (AGUD438; approved date 31 March 2019) and all animal experiments were conducted following the national guidelines and the relevant national laws on the protection of animals. Ultrapure lipopolysaccharide from P. gingivalis was purchased for experiments (InvivoGen, San Diego, CA, USA).

NSC‐34 cells were purchased from Cedarlane (Burlington, NC, USA) (cat. no. CLU140) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin antibiotics (pen/strep). This is the normal growth medium for NSC‐34 cells. For differentiation, cells were harvested using trypsin/EDTA, and cell pellet was washed twice with differentiation medium before seeding into collagen‐coated culture plates (Corning BioCoat, Corning, NY, USA; cat. no. 354400). Four kinds of differentiation media were used in this study: (a) ‘MEM’ – minimum essential medium α (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. 12571063), (b) ‘MEM with atRA’ – MEM with 1 μm all‐trans‐retinoic acid (Sigma‐Aldrich, St Louis, MO, USA; cat. no. R2625), (c) ‘F12’ – 1 : 1 of DMEM and Ham's F12 (Corning Cellgro cat. no. 10‐090‐CV), and (d) ‘F12 with atRA’ – F12 medium with 1 μm all‐trans‐retinoic acid. All differentiation media were supplemented with 1% FBS, non‐essential amino acids (Thermo Fisher Scientific, cat. no. 11140050), and pen/strep. Cells were incubated in a tissue culture incubator at 37 °C in a humidified incubator containing 5% CO₂.

As described previously, 66cl4 cells were stably transduced to overexpress NPNT or NPNT mutants (RGE or RGE‐AIA), while 66cl4 empty vector cells (EV) were used as a control 14. Furthermore, shRNA was used to knock down NPNT protein levels in 4T1 cells (sh‐NPNT), while a nontargeting shRNA in 4T1 cells was used as a control (sh‐ctr) 14. All cell lines were cultured in (1X) minimum essential medium α (Thermo Fisher Scientific, Cat: 22561021), supplemented with 10% fetal bovine serum, 1% (v/v) penicillin–streptomycin, and 1M hepes buffer (Thermo Fisher Scientific, Cat: 15630080). Cell lines were routinely tested for mycoplasma infection.

Human hepatic cancer cells (Hep G2 cells; RCB1642, Riken Cell Bank, Japan), red fluorescent protein-expressing human neonatal dermal fibroblasts (RFP-HNDFCs; cAP-0008RFP, Angio-Proteomie, USA), and normal human hepatocytes (SA152, KaLy-Cell, France) were purchased. Hep G2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Japan) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Japan) and 1% penicillin/streptomycin (Life Technologies, USA). RFP-HNDFCs were maintained in minimum essential medium α (Thermo Fisher, Japan) supplemented with 10% FBS and 1% penicillin/streptomycin. The culture media were exchanged every two days and the cells were passaged with 0.25% trypsin-EDTA (Thermo Fisher, Japan). When Hep G2 cells were mixed with the other types of cells (HNDFCs or normal hepatocytes), we used the culture medium for Hep G2 cells.

MC38 murine CRC cells were obtained from Dr. Eui-Cheol Shin (KAIST, Daejeon, Korea). LLC1 murine lung cancer cells and NK-92 human NK cells were purchased from the American Type Culture Collection (Manassas, VA, United States). HCT116 and KM12SM human CRC cells, CT-26 murine CRC cells, and NCI-H460 human lung cancer cells were purchased from the Korean Cell Bank (Seoul, Korea). MC38 and LLC1 cells were grown in DMEM, and HCT116 cells, KM12SM, CT-26, NCI-H460 cells were grown in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere containing 5% CO₂. NK-92 cells were maintained in Minimum Essential Medium α (Gibco, New York, NY, United States) supplemented with 12.5% horse serum, 12.5% FBS, 0.2 mM myo-inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, penicillin 100 U/ml, streptomycin 100 μg/ml, and 100 U/ml recombinant human interleukin 2 (IL-2, Rocky Hill, NJ, United States). NK-92 cells were subcultured every 2 or 3 days, depending on the cell density.

For DNA extraction from swabs and tissue samples, approximately 25 mg of tissue was ground in 2 mL of a serum-free minimum essential medium α (Gibco, UK) solution by using a homogenizer, and prepared by centrifuging the whole sample at 3500 rpm for 10 min [10 (link)].
DNA was extracted from homogenized clinical samples using the Intron^® Patho Gene-spin™ DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) according to the manufacturer’s instructions. For detection of EHV-1, ORF33 specific real-time PCR was performed [9 (link)]. Two microliters of the DNA extract were applied in the PCRs.