Gibco dulbecco s modified eagle medium dmem

Both MARC-145 cells and human embryonic kidney 293FT cells were cultured in GIBCO Dulbecco’s modified Eagle medium DMEM (Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO₂ atmosphere at 37 °C. Porcine pulmonary alveolar macrophages (PAMs) were prepared as previously described [59 (link)]. PAMs and 3D4/21 cells (ATCC CRL-2843), a PAM-derived cell line, were both maintained in RPIM-1640 (Fisher Scientific) containing 10% FBS. The stock of PRRSV strain JXwn06 with a titer of 10⁷ TCID₅₀/ml was used in this study [60 (link)]. Mouse anti-HA monoclonal antibody (mAb) (H3663), mouse anti-β-actin mAb (A5441), rabbit anti-Myc polyclonal antibody (C3956), rabbit anti-GFP polyclonal antibody (G1544) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-ILF2 mAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-ILF2 polyclonal antibody and rabbit anti-ILF3 polyclonal antibody were purchased from Proteintech Group (Chicago, IL, USA). The mAbs specific for the nsp9, nsp2 and N protein of PRRSV were prepared in our laboratory.

Preparation of PAMs from piglets was performed as previously described [42 (link)]. PAMs were then maintained in RPMI-1640 medium (Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (FBS, Hyclone Laboratories, Inc., South Logan, UT). The African green monkey kidney epithelial cell line MARC-145 cells (ATCC) [43 (link)], and human embryonic kidney (HEK) 293 cells [44 (link)] and 293FT cells (Cell Resource Center, Institute of Basic Medical Science, CAMS/PUMC) [45 (link)] were cultured with GIBCO Dulbecco’s modified Eagle medium (DMEM) (Fisher Scientific) with 10% FBS in a 37°C incubator with 5% CO₂. The PRRSV (JXwn06) used in this study was characterized in our laboratory [46 (link)]. For virus infection assays, MARC-145 cells were cultured to approximately 90% confluence and infected with JXwn06 at a multiplicity of infection (MOI) of 0.01, and then maintained in the medium containing 5% FBS at 37°C until collection.

The compound cisplatin (CDDP, #479306; ≥99.9% purity) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Stock solution of 1 mM dissolved in dimethyl sulfoxide (DMSO, #W387520; Sigma-Aldrich Co., St. Louis, MO, USA) was stored at −20 °C until use. CDDP was dissolved in DMSO and diluted further to desired concentration in sterile complete culture medium immediately prior to use. Propidium iodide (PI, #81845), phosphate buffered saline (PBS, #P7059), sulforhodamine B (SRB, #230162) dye, TRIS base (#93352), and acetic acid (#A6283) were all purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA), Gibco^® Dulbecco’s modified Eagle medium (DMEM, #11960077), fetal bovine serum (FBS, #A31605), and trypsin-EDTA (#25300054) were purchased from Thermo Fisher (Thermo Fisher Scientific Inc., Waltham, MA, USA). The QIAGEN OneStep RT-PCR kit (#210212) was obtained from Qiagen (QIAGEN Inc., Germantown, MD, USA). BD Pharmingen™ FITC Annexin-V apoptosis detection kit I (#556547) was from BD (BD Biosciences, San Jose, CA, USA).

HEK293T and C2C12 cells were cultured in Gibco Dulbecco’s modified Eagle medium (DMEM) (Gibco, Co Dublin, Ireland) supplemented with 10% FBS in a humidified incubator with 5% CO₂ at 37 °C. To induce myogenic differentiation, C2C12 cells were incubated in DMEM supplemented with 2% heat-inactivated horse serum (Gibco).

Both HeLa and HEK293T cells were cultured in GIBCO Dulbecco’s modified Eagle medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) at 37°C in a humidified 5% CO₂ atmosphere. Cells (2.5 × 10⁵ cells/well) were seeded in a 12-well plate or 100-mm dishes. After 24 h, plasmids, siRNA, or poly(I:C) (InvivoGen, USA) was transfected into HEK293T and HeLa cells with Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the manufacturer’s recommendations. The sequence of the siRNA specific for HSP90B1 was 5’-CTTCGCCTCAGTTTGAACA-3’. Both siRNA-HSP90B1 and siRNA-NC (5 nmol, siN0000001-1-5) were purchased from RiboBio (China).