293t cells

293T cells were obtained from the Riken BioResource Center Cell Bank. Human bronchial epithelial (Calu-3) cells and chicken fibroblast (DF-1) cells were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS). AI viruses A/duck/Egypt/D1Br/2007 (denoted D1 here) and A/chicken Egypt/CL69/2013 (denoted EG13 here) are representative ancestral and contemporary strains of H5N1 clade 2.2.1, respectively. The details of the two viruses were described previously (6 (link), 24 (link), 63 (link)). All recombinant H5N1 viruses were propagated once in 9-day-old embryonated eggs and purified by ultracentrifugation as described previously (24 (link)).

A549 cells (human lung adenocarcinoma cell line), THP-1 cells (human acute monocytic leukemia cell line), RAW 264.7 cells (mouse macrophage cell line), and 293T cells were obtained from the Bioresource Collection and Research Center, Taiwan. The A549 cells and THP-1 cells were cultured in RPMI-1640 medium (Invitrogen Corp., Carlsbad, CA, USA), supplemented with 10% fetal bovine serum at 37 °C and 5% CO₂. The 293T cells and RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA, USA), supplemented with 10% fetal bovine serum at 37 °C and 5% CO₂. The indicated cells were cultured to 60–70% confluence before treatment. Then, the medium was then replaced with a fresh medium containing the indicated compounds at the indicated concentrations. The cells treated with water alone were used as untreated vehicle controls.

Briefly, 293T cells (RIKEN BioResource Center, Tsukuba, Japan) were cultured in the Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Pure Chemical) supplemented with heat-inactivated 10% fetal bovine serum (GE Healthcare, Buckinghamshire, UK), nonessential amino acids (Thermo Fisher Scientific, Waltham, MA, USA), and an antibiotic-antimycotic solution (100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B; Thermo Fisher Scientific). The cells were seeded in 96-well plates (BD Biosciences, Heidelberg, Germany) at a density of 1 × 10⁴ cells/well. At 24 h after the seeding, the cells were transfected with plasmids.

We used HPC patient-derived xenograft cell lines (HPCM1^{7 (link),8 (link)} and HPCM2^{7 (link),8 (link)} cells), which were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 unit/mL penicillin, and 100 μg/mL streptomycin. MCC148c cells^{13 (link)}, established by patient-derived xenografts of cancer tissue from a lung squamous cell carcinoma patient^{5 (link)}, were maintained in DMEM supplemented with 10% FBS, 0.4 mg/mL hydrocortisone, 2.5 mM Y-27632 (Focus Biomolecules, Plymouth, PA, USA), and penicillin/streptomycin. DMEM supplemented with 10% FBS and penicillin/streptomycin was used to maintain 293 T cells (RIKEN BioResource Center, Kyoto, Japan). Het-1A cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in airway epithelial cell basal medium from the airway epithelial cell growth medium supplement pack (PromoCell, Heidelberg, Germany) supplemented with 4% FBS and penicillin/streptomycin. IMR-32 cells were purchased from ATCC and maintained in DMEM supplemented with 10% FBS and 0.1 mM non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA). HSC-3 cells were purchased from RIKEN (Saitama, Japan) and maintained in Eagle’s minimal essential medium supplemented with 10% FBS and penicillin/streptomycin.

Human prostate cancer cells (PC-3, DU-145 and LNCaP) and 293T cells were obtained from RIKEN Bio Resource Center. Other human prostate cancer cells (22Rv1 and RWPE-1) were obtained from American Type Culture Collection (ATCC). PC-3M (human prostate cancer cell lines) and normal prostate cell lines (PNT2) were generous gifts from Dr. Youqiang Ke (Liverpool University, UK). All cell lines were male and cultured at 37°C in a humidified atmosphere (5% CO₂). All cell lines, except for 293T, were grown in RPMI 1640 medium (Thermo Scientific) supplemented with 10% (v/v) FBS (Thermo Scientific). The 293T cell line was grown in DMEM/high glucose (Thermo Scientific) supplemented with 10% (v/v) FBS (Thermo Scientific). All cells tested negative for mycoplasma.

293T cells (human embryonic kidney cell line) and Vero cells (African green monkey kidney cell line) were obtained from the RIKEN BioResource Center Cell Bank. Calu-3 cells (human bronchial epithelial cell line), MRC5 cells (human fetal lung fibroblast cell line) and HCT8 cells (human rectal adenocarcinoma cell line) were obtained from the American Type Culture Collection (ATCC). THP-1 cells and Vero/TMPRSS2 cells⁵³ (link) were obtained from the Japanese Collection of Research Bioresources Cell Bank. 293T, Calu-3 and MRC5 cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS). HCT8 and THP-1 cells were maintained in RPMI-1640 medium with 10% FCS. Vero cells and Vero/TMPRSS2 cells were maintained in DMEM containing 10% FCS. 1 mg/mL G418 (Invivogen) was added to the growth medium for Vero/TMPRSS2 cells. THP-1-dual reporter cells, THP-1-dual KO-RIG-I cells and THP-1-dual KO-MDA5 cells were purchased from InvivoGen and maintained according to the manufacturer’s instructions.

Jurkat E6.1 cells (European Collection of Cell Cultures, Salisbury, UK), K562 (RIKEN BioResource Center, Ibaraki, Japan), and CD19/K562 cells that were generated by transduction of K562 cells with human CD19-expressing retrovirus vectors were grown in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S) (RPMI complete medium). 293T cells (RIKEN BioResource Center) were cultured in DMEM/F-12 medium (Life Technologies) supplemented with 10% FBS and P/S (DMEM/F-12 complete medium). All cultures were maintained in an incubator at 37°C with 5% CO₂.