Dulbecco modified eagle medium (dmem)

The human adenocarcinoma alveolar epithelial cell line A549 and RSV were kindly offered by Jiangsu Key Laboratory of Pediatric Respiratory Disease. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Basal Media, China) added with 10% fetal bovine serum (FBS, Life-iLab, China), 100 μg/ml streptomycin and 100 U/mL penicillin (Basal Media, China) under 37°C with a 5% CO2 environment. In some experiments, cells were pretreated for 2 h with AC, and then inoculated with RSV (multiplicity of infection [MOI] of 1).

The porcine IPI-2I intestinal epithelial cells were propagated in DMEM (Basal Media, Shanghai, China) containing 10 % fetal bovine serum. The cells were seeded into 24-well plates at a concentration of 4×10⁵ cells per well, and cultured overnight at 37 °C in 5 % CO₂. The S. Derby S35 WT and Δdam strains were grown until stationary phase at 37 °C in LB medium, and subsequently added to each well with a Multiplicity of infection (MOI) of 20 : 1. The cell cultures were incubated at 37 °C in 5 % CO₂ for 1 h, after which the cultured cells were washed with DPBS (Gibco, Grand Island, NY, USA) three times and solubilized with 1 % Triton X-100 for 5 min; finally, the adhered bacterial cells were counted. For invasion, 100 µg ml⁻¹ of gentamycin was added to the cell cultures to lyse the extracellular bacteria after washing, before culturing them for an additional 1 h at 37 °C in 5 % CO₂. After washing and solubilizing, the solution was serially diluted and the appropriate dilutions were coated onto the LB plates to calculate the number of bacteria. Data were analysed using the two-tailed Student’s t-test.

LCMV Armstrong 53b strain, BHK-21, and Vero cells were a generous gift of Prof. Ye (Chongqing). The virus was grown on BHK-21 cells and quantitated by plaque assay on Vero cells as described elsewhere (20 (link)). For acute infections, mice were injected with 2•10⁵ PFU of LCMV Armstrong i.p.
To determine LCMV titers in the spleen and liver, harvested organs were weighed, weight-adjusted to 1/10 with DMEM (Basal Media, Shanghai) with 2% FBS (Gibco) and 1% penicillin/streptomycin solution (Hangzhou Keyi Biotechnology Co., Ltd). Tissue samples were homogenized with metal beads at the Tissue Lyser 48 (Shanghai Jing Xin Industrial Development Co., Ltd) and clarified by centrifugation at 1,000 g for 10 min. Then the samples were frozen at −80°C and analyzed later. Organ homogenates were diluted in 10-fold increments and tested in the standard 4-day plaque assays.

Cardiac fibroblasts (CFs) were isolated from neonatal mice, as previously described.^[65] Neonatal mouse CFs were cultured in DMEM (Basal Media, Shanghai, China) with 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 1% penicillin/streptomycin at 37°C under 5% CO2 in a humidified incubator.

Human embryonic kidney epithelial cell line (293T) and human cervical cancer cells (HeLa) were grown in Dulbecco’s modified Eagle’s medium (DMEM, BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China) and 1% penicillin/streptomycin (Gibco, Gaithersburg, MD, USA) under standard tissue-culture conditions (37 °C, 5% CO₂). HeLa cells stably overexpressing GFP-G3BP1 or GFP-G3BP2 were grown in DMEM with 10% FBS and 2 μg/mL of puromycin [24 (link)]. The cells were seeded in multiwell plates or dishes and transfected with plasmids at approximately 60~70% confluence using PEI (Polyscience, Niles, IL, USA) or lipofectamine 2000 (ThermoFisher, Waltham, MA, USA).

Mouse L929 cells were seeded into 96-well tissue culture plates at a density of 1.5×10⁴ cells/well in DMEM (Basalmedia) supplemented with 3% FBS (Moregate). The cells were incubated at 37°C for 24 h and then incubated with gradient concentrations of TNFα antagonists in the presence of 1 μg/ml actinomycin D (Solarbio) and 20 IU/ml sTNFα (NIBSC, 12/154) for 18 h at 37°C. Cells without TNFα antagonists and sTNFα treatment served as controls. Cell viability was measured by MTS assay (Promega). In the MTS test, the cells in each well were incubated with MTS reagent mixture for 4 h, and MTS absorbencies were measured at 490 nm using a microplate reader SpectraMax M2^e (Molecular Devices). Percentage cell viability was calculated by dividing the absorbance values of drug treated wells by those of control wells and multiplying by 100. The dose response curve was fitted with a 4-parameter model using GraphPad Prism 5 software (GraphPad Software).