Pgl4.74 vector

Luciferase reporter plasmids, pGL3-VEGF promoter constructs containing four copies of ERRE site, (5′-AGGTCA-3′), were kindly provided by Prof. Salman Hyder (University of Missouri, Columbia, MO, USA). pGL4.74 vector (Promega, Madison, WI, USA) was used to normalize the luciferase activities.

The 3′ UTR was fuesed to downstream of the Renilla luciferase stop codon in pGL4.74 vector (Promega, USA). To generate the mutant 3′ UTR of human IL-21, two-step PCR mutagenesis was performed^{51 (link)} using the WT-3′ UTR as a template. 293 T cells cultured in 96-well plate were co-transfected with the reporter vector (Renilla or Renilla-hIL-21 3′ UTR) (2 ng), a transfection control vector expressing firefly luciferase (pGL3-control) (20 ng), and negative control RNA (NC) or miRNA (Bioneer) using Polyethylenimine. Cells were harvested at 24 hours post-transfection and assayed with Dual Luciferase Assay (Promega). Three independent experiments were performed with a triplicate set.

Jurkat cells (10⁷/ml) were transiently transfected with the IL-2-Luc, TNF-Luc and IL-17-Luc plasmids using the Lipofectine™ reagent (Life Technologies; Madrid, Spain), according to the manufacturer's recommendations. Twenty-four hours after transfection, the cells were pre-treated with VCE-003 for 30 min and stimulated for 6 h with the OKT3 (coated 1 µg/ml) and the anti-CD28 antibody 15E8 (0.5 µg/ml). The cells were then lysed in 25 mM Tris-phosphate buffer [pH 7.8] containing 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100, and 7% glycerol. The luciferase activity was quantified on an Autolumat LB 953 (EG&G Berthold; Oak Ridge, TN, USA), following the manufacturer's instructions (Luciferase assay kit, Promega; Madison, WI, USA), and the protein concentration was measured by the Bradford method. The pGL4.74 vector (Promega; Madison, WI, USA) that contains a constitutively expressed firefly luciferase gene served as an internal control to normalize transfection efficiency and firefly and renilla luciferase activities were measured using the Dual-Luciferase® reporter assay system (Promega; Madison, WI, USA). The background obtained with the lysis buffer was subtracted from each experimental value and the specific transactivation was expressed as the fold induction relative to the untreated cells. All the experiments were repeated at least three times.