Penicillin and streptomycin

Four EGFR‐mutant lung adenocarcinoma cell lines were used in this study. HCC827 cells (with a deletion in exon 19) were purchased from the American Type Culture Collection and PC‐9 cells (with a deletion in exon 19) were obtained from Immuno‐Biological Laboratories. These cells were obtained from 2010 to 2016. Osimertinib‐resistant HCC827 (HCC827‐OR) and osimertinib‐resistant PC‐9 (PC‐9‐OR) cell lines were established using a stepwise method, as previously described.¹⁷ Cells were amplified and frozen, and one aliquot of each was thawed for this research. All cells were routinely screened for the absence of mycoplasma and cultured in RMPI 1640 medium (FUJIFILM Wako Pure Chemical Co.) with 10% heat‐inactivated fetal bovine serum (FBS) (BioWest) and 1% penicillin and streptomycin (FUJIFILM Wako Pure Chemical Co) at 37°C in a 5% CO₂ incubator. Falcon Cell Culture Inserts (Corning Inc.) with a transparent polyethylene terephthalate membrane (pore size: 0.4 μm) were used for co‐culture. HCC827 and PC‐9 cells were plated into six‐well plates (2 × 10⁵ cells/well), the device was placed on the six‐well plates, and equal numbers of their respective resistant cells were plated into the device. After 72 h, the device was removed and HCC827 and PC‐9 cells were used for protein extraction.

The human fetal lung fibroblast cell line, TIG-3-20, was purchased from JCBR cell bank, National Institutes of Biomedical Innovation, Health, and Nutrition. TIG-3-20 cells were cultured in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) containing 10% fetal bovine serum (173012, Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL penicillin and streptomycin (Wako) at 37 °C in a humidified atmosphere with 5% CO₂. TIG-3-20 cells were used between passage 30 and 40.

BMCs were isolated from WT mice and were incubated in RPMI1640 culture media (Thermo Fisher Scientific) containing 10% FBS (Sigma Aldrich, St. Louis, MO, USA) and 50 U/mL of penicillin and streptomycin (FUJIFILM Wako Pure Chemical) overnight. When necessary, the BMCs were treated with 50 µmol/L Asc or 200 nmol/L dexamethasone (FUJIFILM Wako Pure Chemical) 2 h before X-irradiation. One day, following the exposure to X-rays, the medium containing BMCs was collected and centrifuged at 15,000× g for 5 min at 4 °C. The supernatant was subjected to Cytotoxicity LDH Assay Kit-WST (Dojindo, Kumamoto, Japan) in accordance with the manufacturer’s instructions.

Primary HUVECs (C2519A, Lonza, Basel, Switzerland) were cultured at 37 °C in EGM-2 supplemented with 2% fetal bovine serum (Lonza). All plates and coverslips used for HUVECs were freshly coated with fibronectin from human plasma (20 μg/mL; Fujifilm Wako, Osaka, Japan). Lenti-X293T cells (Clontech) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Fujifilm Wako) supplemented with 10% EqualFETAL (Atlas Biologicals, CO, USA) and 1 μg/mL penicillin and streptomycin (Fujifilm Wako) at 37 °C.

Akata-LCL was established from human umbilical cord blood B cells infected with Akata strain of EBVs. X50-7, established from human umbilical cord blood lymphocytes infected by B95-8 strain of EBVs^{46 (link)}, was kindly provided. All cell lines were cultured in RPMI1640 medium (Wako) supplemented with 10% fetal bovine serum (Hyclone), 1% sodium pyruvate (Wako), 1% penicillin and streptomycin (Wako), 1% non-essential amino acids (Wako), and 50 μM 2-mercaptoethanol (Gibco). The cells were incubated in 37 °C humid atmospheres, and 1/4–1/3 volume of the cells were passaged twice a week.

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.