Lipofectamine 2000 lipo2000

Sodium butyrate, propionate, TSA, WST-1, and a protease inhibitor cocktail were purchased from Sigma-Aldrich. Butyrate and propionate were dissolved in endothelial growth factor (EGM-2) and TSA in DMSO and then further diluted in EGM-2. Concentrations used were based on recent publications of our group [5 (link),6 (link)]. Human recombinant TNFα was bought from eBioscience. The human IL-8 enzyme-linked immunosorbent assay (ELISA) kit, Lipofectamine 2000 (lipo-2000), and the BLOCK-iT Alexa Fluor Red Fluorescent control were purchased from Invitrogen. The NF-κB p65 (pS536) ELISA kit was bought from Cell signaling technology. The IL-33 ELISA kit was purchased from U-CyTech biosciences. The IL-33 monoclonal antibody and Sliencer GAPDH siRNA (human) were bought from Thermo Fisher Scientific. Silencer pre-designed on-Targetplus SMARTpool siRNA IL-33 and siGENOME Non-Targeting siRNA pool (Silencer negative control) were bought from Dharmacon. The following monoclonal antibodies: the anti-phospho p38 (phospho T180 + Y182) antibody, the anti-JNK1+JNK2 (phospho T183 + Y185) antibody, the anti-ERK1/2 (phospho Thr202/Tyr204) antibody, the anti-GAPDH antibody, the rabbit anti-mouse IgG H&L (HRP) conjugated antibody, and the goat anti-rabbit IgG H&L (HRP) conjugated antibody were purchased from Cell Signaling Technology and Abcam.

HEK 293T cells and Vero cells were grown in Dulbecco’s modified Eagle medium (DMEM) (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin and streptomycin. CIK (Ctenopharyngodon idellus kidney) cells were grown in minimum essential medium (MEM) (Gibco-BRL) supplemented with 10% FBS respectively. GCRV-873 isolated and stored in the author’s laboratory was propagated in CIK cells as described previously [34 ].
GCRV VP1, VP4, VP6, VP7, NS38 and NS80 antibodies were generated and stored in our laboratory [31 (link), 35 (link)–38 (link)]. Mouse anti-Flag monoclonal antibody (mAb) was purchased from Abmart (Shanghai, China). Mouse monoclonal IgG2b isotype control antibody was purchased from eBioscience Inc. (San Diego, CA). Mouse anti-β-actin mAb, rabbit anti-poly-ubiquitin and anti-vimentin polyclonal antibodies (pAbs) were purchased from Proteintech (Wuhan, China). Alexa Fluor^® 488 or 568 donkey anti-rabbit IgG (H+L) antibody, Alexa Fluor^® 488 or 568 donkey anti-mouse IgG (H+L) antibody and Lipofectamine 2000 (Lipo2000) were purchased from Invitrogen Co. (Invitrogen, Carlsbad, USA).

N-Fluorenyl-9-methoxycarbonyl (Fmoc)-protected L-type amino acids and D-type amino acids were purchased from GL Biochem Ltd. and CS Bio Ltd. (Shanghai, China), respectively. Rink amide resin (0.44 mmol g⁻¹) was purchased from Nankai Hecheng (Tianjin, China). 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexauorophosphate (HBTU) and hydroxybenzotriazole (HOBT) were purchased from GL Biochem Ltd. Diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), thioanisole, ethandithiol, and anisole were purchased from J & K Scientific (Beijing, China). All other solvents were redistilled from drying reagents.
Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (CA, USA). Penicillin–streptomycin, phosphate-buffered saline (PBS), YOYO-1 (491/501) intercalating dye, and Lipofectamine 2000 (Lipo2000) were purchased from Invitrogen (CA, USA). A pGL3 control vector, Dual-Glo® Luciferase Assay System, and CellTiter 96® Aqueous One Solution Cell Proliferation Assay were purchased from Promega (WI, USA). A Bradford protein assay kit was purchased from Beijing Solarbio Science & Technology Co. Ltd. (Beijing, China).

Human embryonic kidney cell line (HEK293T) and a human cervical cancer cell line (HeLa) were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin. HTLV-1–transformed cell lines MT2 and MT4 were described previously (42 (link)). Jurkat, MT2, and MT4 cells were grown in RPMI 1640 medium containing 1% penicillin-streptomycin and supplemented with 10% FBS. All cells were cultured at 37°C in a 5% CO₂ incubator.
To generate stable Jurkat-shNC, Jurkat-shPCBP1, MT2-shNC, MT2-shPCBP1, MT4-shNC, MT4-shPCBP1, HEK293T-shNC, and HEK293T-shPCBP1 cell lines, HEK293T cells were seeded into 6-well plate and transfected with 2 μg of PLKO.1 vector encoding shNC or shPCBP1, 1.5 μg of psPAX2, and 0.5 μg of pMD2.G plasmids with Lipofectamine 2000 (Lipo2000) (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. At 24 h and 48 h after transfection, the lentivirus was collected from HEK293T supernatants. After incubation with the lentivirus for 48 h, the Jurkat, MT2, MT4, and HEK293T cells were selected by puromycin (2.5 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), and the knockdown efficiencies of shRNAs were determined by Western blotting.

Small interfering RNA (siRNA) was synthesized by Biomics Biotechnology Co., Ltd. (Nantong, China). The siRNA sequences are listed in Table S2. Fibroblasts in complete DMEM were seeded in six-well plates (2×10⁵cells) and cultured at 37°C under 5% O₂, 5% CO₂ and 90% N₂. At 70–80% confluence, cells were incubated for 6 h in complete medium free of penicillin and streptomycin before transfection for a further 6 h with 50 nM siRNA using lipofectamine 2000 (lipo2000, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Subsequently, the fibroblasts were cultured in complete DMEM. The efficiency of siRNA-mediated silencing was confirmed by real-time PCR analysis of mRNA expression levels of target genes (STMN1, JIP1, and JNK3) after transfection for 24 h and western blot analysis of protein expression levels after 48 h.