Gibco dmem

Human HEK293T cells were maintained in Gibco™ DMEM with 10% FBS and 1% penicillin/streptomycin (ThermoFisher Scientific, MA, USA). Empty pBABE vector and/or plasmids encoding RET wild type or mutant alleles were transiently transfected using Lipofectamine 3000 (ThermoFisher Scientific, MA, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were trypsinized and plated into 100-mm plates. Transfected cells were selected with 0.75 μg/ml puromycin for 1 week until all cells in the control plates were dead. Then cells were trypsinized, counted and 5000 cells were plated into 60-mm plates. Cells were kept in DMEM with 10% bovine serum. The culture medium was changed every 3–4 days. Approximately 10 days after transfection, cells were stained by crystal violet. The colony formation experiments were repeated three times.

The experiments were carried out using a previously established M. myotis cell line [40 (link)] derived from the olfactory nerve (MmNOl cells). The bat cell cultures were cultivated in $\text{Gibco}$ DMEM, High Glucose, $\text{GlutaMAX}$ medium (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS) supplement (Thermo Fisher Scientific) at 37  ${}^{\circ }$ C with 5% CO ${}_2$ . The cell-adapted EBLV-1 strain 8918FRA [64 (link)] was used, and virus stock was prepared on BSR cells.
The cells were cultivated in two groups; at 37  ${}^{\circ }$ C, to simulate euthermia and at 5  ${}^{\circ }$ C to simulate torpor. Each of the temperature groups contained an infected sample and a non-infected control (Fig. 1). The experimental design thus consisted of cells under four experimental conditions. All of them were inoculated in independent laboratory session triplicates to prevent technical errors in the results.
An amount of $2.5\times {10}^6$ cells was seeded on T25 flask. At 18 h after seeding, the cells were infected with lyssavirus EBLV-1 8918FRA at multiplicity of infection (MOI) 1 (i.e., $2.5\times {10}^6$ fluorescent focus-forming unit (FFU) in 700  $\mu$ L of medium without FBS. After 1 h, the cell culture medium was replaced with a fresh, virus-free medium containing 10% FBS and the cells were incubated for 6 h at 37  ${}^{\circ }$ C with 5% CO ${}_2$ . Then, the cells were incubated 48 h at 37  ${}^{\circ }$ C with 5% CO ${}_2$ or at 5  ${}^{\circ }$ C after closing the flasks.

Human (Homo sapiens) adenocarcinoma alveolar basal epithelial A549 (American Type Culture Collection [ATCC], Manassas, VA; #CCL-185) cells, epithelial Hela cells (ATCC, #CCL-2), and hepatocarcinoma Huh-7 cells (a kind gift from Hideki Ebihara, Rocky Mountain Laboratories, Hamilton, MT, USA), and grivet (Chlorocebus aethiops) kidney epithelial Vero cells (ATCC, #CCL-81), and Vero E6 cells (ATCC, #CRL-1586) were grown in Gibco Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA). Syrian golden hamster (Mesocricetus auratus) kidney BHK-21 fibroblasts (ATCC, #CCL-10) were grown in Gibco DMEM supplemented with 10% FBS and 5% tryptose phosphate broth (TPB, Thermo Fisher Scientific). All cells were incubated at 37°C in a humidified 5% CO2 atmosphere.

The RAW264.7 macrophage cell line (China Infrastructure of Cell Line Resource, Beijing, China) was used to evaluate the effect of saponins on cholesterol metabolism. The cells were incubated in the Gibco® DMEM, containing 10% Gibco® fetal bovine serum (North America, Thermo Fisher Scientific Inc., Waltham, USA), maintained at 37 °C under a humidified 5% CO₂ environment. The homogeneous cell suspension (1 × 10⁵/mL) were cultured in a 12 well-culture plate for 24 h. The plate was treated with different fractions of Thelenota ananas saponins (filtration by 0.45 μm filter membrane) for 24 h at 37 °C. The control group was treated with an equal volume of serum-free culture medium. Then, the supernatant was collected and used to detect the content of LDL-C via the LDL-C assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).