Pcmv β gal plasmid

To examine the effect of PB on the activation of the Il17 promoter, Jurkat cells were co-transfected with pCMV-β-Gal plasmid (Clontech, Mountain View, CA), pCMV10-3xFlag-RORγ plasmid, and a pGL4.14 reporter plasmid (Promega Corp., Madison, WI, USA) under the control of human Il17-3kb-CNS promoter^{46 (link)}, and then treated with vehicle, or PB (0.5μmol/l), or GW9662 (1.0 μmol/l) for 1 h before PB incubation. After 24 h, cell extracts were lysed and the firefly luciferase and β-galactocidase activities were measured using a Luciferase Assay Substrate kit (Promega) and a Luminescent β-galactosidase Detection kit II (Clontech), respectively.

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)).