Polyethyleneimine reagent

293 T cells and HeLa cells were obtained from the American Type Culture Collection. Plasmids were transfected to 293 T cells by using the polyethyleneimine reagent (Polysciences) and transfected to HeLa cells by using Entranster™-D (Engreen Biosystem). For the inhibition of proteasome, 5 μM MG132 (Sigma-Aldrich) was added into the culture medium and cells were incubated for additional 8 hours unless indicated otherwise.

C2C12 and KS483 cells were split at a density of 1.5 × 10⁴ cells per cm² in 12-well plates. The following day, cells were transiently transfected with plasmid constructs expressing shRNA targeting IL-6 mRNA or control scrambled shRNA (0.5 µg of total DNA per well). Transfection was carried out using GeneJuice transfection reagent (Merck Millipore, Billerica, MA, USA) following the manufacturer’s protocol. The efficacy of shRNA-mediated knockdown was confirmed by quantitative polymerase chain reaction (PCR) and varied from 6.5 to 8 times for most efficient variants (data not shown).
For luciferase reporter assays, NIH-3T3 cells were split at a density of 1.5 × 10⁴ cells per cm² in 12-well plates. Transfection was carried out using polyethyleneimine reagent (Polysciences, Warrington, PA, USA). pcDNA3 plasmid (Invitrogen) was used as a control vehicle. Co-transfection with pCMV-β-Gal plasmid (Clontech-Takara, Mountain View, CA, USA) was used as an internal control for the efficacy of transient transfection. β-Galactosidase activity in cellular lysates was quantified spectrophotometrcally in 100 mmNa₂HPO₄/NaH₂PO₄, 1 mm MgCl₂, 100 mm 2-mercaptoethanol, and 0.67 mg/mL O-nitrophenylgalactopyranoside essentially as before (21 (link)).

An engineered pTT5 plasmid with a TEV enzyme cleavage site, a human IgG1 Fc at the C terminus, and an IFNA1 signal peptide at the N terminus were used for VNAR-Fc fusion expression. Electro-competent Escherichia coli TG1 cells were preserved in our laboratory.
Human embryonic kidney (HEK) 293F cells were obtained from TJ-Lab. The cells were maintained in Union-293 (Union, Shanghai, China) supplemented with 100 units/mL of penicillin-streptomycin (Gibco, Carlsbad, CA, USA), then cultured at 5% CO₂ and 37 °C. DMEM (VivaCell, Denzlingen, Germany) and polyethyleneimine reagent (PEI, Polyscience, Warrington, PA, USA) were used for cell transfection.