Viromer red reagent

ChIP-seq enhancer peak regions assigned to Sox4, Sox11, Il15 and Mapk1 were amplified from mouse genomic DNA and cloned into pBV- Luc, a luciferase reporter plasmid with a minimal promoter and very low basal activity (24 (link)). These reporter plasmids were co-transfected with 3X-FLAG SOX4 or 3X-FLAG SOX11 expression plasmids (14 (link)) into HEK293 cells using Viromer Red reagent (Origene). pSV2bgal plasmid was used as a control for transfection efficiency. Cells were treated with 10ng/mL recombinant TNF (R&D systems) for the last 16h of the 36h transfection period. Luciferase and β-galactosidase activities were assayed using the Dual-light combined reporter gene assay system (Applied Biosystems) using Synergy H1 Multi-Mode Plate Reader (Biotek). Reporter activities were normalized for transfection efficiency and reported as fold change in luciferase activity. Assays were performed as triplicates.

Cells were harvested and lysed using Pierce™ IP lysis buffer (Thermo Fisher Scientific) containing 1% Halt™ protease and phosphatase inhibitors (Thermo Fisher Scientific). Equal amounts of protein were electrophoresed on 10% SDS–polyacrylamide gels. The primary antibodies used for the western blot are listed in Additional file 1: Table S2. Bands were visualized using ChemiDoc MP Imaging System (Bio-Rad). For luciferase reporter assays plasmids encoding NF-κB (pNL3.2.NF-kB-RE (Promega) and IL-15 [39 (link)], RA SFs were transiently transfected with Viromer Red reagent (Origene). The pSV2bgal plasmid was used as a control for transfection efficiency. Cells were treated with LPS (35 ng/mL), ST2825 (10 μM), or both for 24 h. Luciferase and β-galactosidase activities were assayed using the GloMax Discover System (Promega). Reporter activities were normalized for transfection efficiency and reported as fold change in luciferase activity. Assays were performed as triplicates.