Penicillin

HepG2 cells and their derivatives (NTCP-expressing HepG2 cells [NTCP/HepG2]) were maintained in a Williams’ B culture medium (Gibco)-based PMM (primary hepatocyte maintaining medium), which contained 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Nacalai Tesque, San Diego, CA, USA), 50 µM hydrocortisone (Sigma-Aldrich), 5 µM dexamethasone, 5 µg/mL transferrin (Wako Pure Chemicals), 10 ng/mL EGF (ThermoFisher, Waltham, MA, USA), 5 µg/mL insulin (Sigma-Aldric), 5 ng/mL sodium selenite, 2 mM L-glutamine (Nacalai Tesque), and 0.5 mg/mL G418 (Nacalai Tesque). NTCP-expressing HepG2 cells were the kind gift of Dr. Watashi [43 (link),44 (link)] and were maintained in the same PMM but with the addition of 0.5 mg/mL G418 (Nacalai Tesque). HEK293T cells were cultured in Dulbecco’s modified Eagle media (DMEM) (high glucose) (Nacalai Tesque) supplemented with 10% FBS, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Nacalai Tesque). All the cells were maintained in a 5% CO₂ incubator (Panasonic Health Care, Tokyo, Japan). Huh6 and HB611 cells were maintained in DMEM (low glucose) (Nacalai Tesque) with the same supplements as HEK293T. In the case of HB611, G418 (Nacalai Tesque) was added to the medium at 0.5 mg/mL.

Bone marrow-derived macrophages (BMMs) were prepared as described previously (Ichinohe et al., 2009 (link)). In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque). Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum (FBS), and L-glutamine (2 mM) at 37°C/5% CO₂. HEK293FT cells (a human embryonic kidney cell line) and HeLa cells (a human epithelial carcinoma cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Nacalai Tesque). MDCK cells (Madin-Darby canine kidney cells) and HT-1080 cells (a human fibrosarcoma cell line) were grown in Eagle’s minimal essential medium (E-MEM; Nacalai Tesque) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Nacalai Tesque).
Influenza A virus strain A/PR8 (H1N1) was grown at 35°C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs (Ichinohe et al., 2009 (link)). The viral titer was quantified in a standard plaque assay using MDCK cells (Pang et al., 2013 (link)).

B16F10 murine melanoma cells were purchased from the American Type Culture Collection. B16F10 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque) supplemented with heat inactivated 10% fetal bovine serum (FBS) (Biowest), 100 U/mL penicillin, and 100 µg/mL streptomycin (penicillin–streptomycin Mixed Solution) (Nacalai Tesque). ID8-luc2 murine ovarian cancer cells were a gift from the Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine. ID8 luc2 cells were cultured in Roswell Park Memorial Institute (RPMI) (Nacalai Tesque) supplemented with heat inactivated 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin, at 37 °C and 5% CO₂ in the CO₂ incubator. NHEJ was inhibited by 1 µM NHEJ inhibitor-SCR7 following manufacture’s instructions (Xcess Biosciences Inc.) after electroporation using the NEON system (Thermo Fisher Scientific).

C57BL/6NCrSlc (C57BL/6N) mice were purchased from SLC. Mice lacking IFN-γR have been previously described (30 (link)). All animal experiments were conducted with the approval of the Animal Research Committee of Research Institute for Microbial Diseases at Osaka University. RHΔhxgprtΔku80 and its derivatives of T. gondii were maintained in Vero cells by bi-weekly passage in RPMI (Nacalai Tesque) supplemented with 2% heat-inactivated fetal calf serum (FCS; JRH Bioscience), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Nacalai Tesque). Mouse embryonic fibroblasts were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque) supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Nacalai Tesque).

TC71, SK‐ES‐1, RD‐ES, RD, and human epithelial kidney 293T cell lines were provided by the laboratory of Professor Tohru Sugimoto (University of Miyazaki, Kyoto Prefectural University of Medicine). The SU‐CCS‐1 cell line was kindly supplied by Dr Alan L. Epstein (Keck School of Medicine, University of Southern California), and the HS‐Os‐1 cell line was obtained from Riken Bioresource Center (Tsukuba, Japan). Human fibroblasts were obtained through the culture of mononuclear cells isolated from the bone marrow of healthy volunteer with informed consent. Except for HS‐Os‐1 and 293T, all cells were grown in RPMI (Sigma‐Aldrich) with 10% fetal bovine serum (Sigma‐Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (Nacalai Tesque Inc). HS‐Os‐1 and 293T cells were maintained in Dulbecco's Modified Eagle's Medium (Sigma‐Aldrich) with 10% fetal bovine serum (Sigma‐Aldrich), 100 U/mL penicillin and 100 µg/mL streptomycin (Nacalai Tesque Inc). SK‐ES‐1 and RD‐ES were cultured on collagen‐coated dishes or flasks.

The human cell lines A549, ACHN, ADR-RES, H23, OVCAR-3, OVCAR-4, OVCAR-5, SK-OV3, SK-MEL28, and T-47D were obtained from Dr. Toru Okamoto (the Walter & Eliza Hall Institute). Mouse fibroblast L929, mouse embryonic fibroblast (MEF), baby hamster kidney BHK, African green monkey kidney cell lines (Vero, CV-1, and MA104), canine kidney MDCK, and quail fibrosarcoma QT-6 cell lines were obtained from the American Type Culture Collection. Bovine kidney epithelial MDBK cell line and swine kidney SK-6 cell line [69 (link)] were kindly provided from Dr. Tomokazu Tamura. These human and animal cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 5% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque). BHK-T7 cells were cultured in DMEM supplemented with 5% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 8 μg/mL puromycin (Sigma-Aldrich) [70 (link)]. DemKT1 and YubFKT1 cell lines were kindly provided from Dr. Ken Maeda [49 (link)] and grown in Roswell Park Memorial Institute 1640 medium (RPMI; Nacalai Tesque) supplemented with 5% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Wild-type rsMB was generated previously using a reverse genetics system [45 (link)]. Viruses were propagated in L929 cells. Viral titers were determined by plaque assay using L929 monolayers.