Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

Chad N. Brocker; Donghwan Kim; Tisha Melia; Kritika Karri; Thomas J. Velenosi; Shogo Takahashi; Jessica A. Bonzo; David J. Waxman; Frank J. Gonzalez

for PPRE reporter constructs (Promega, Madison, WI) using a BioRad Quick Ligation Kit (BioRad, Hercules, CA, USA). Prior to performing assays, all constructs were confirmed by Sanger sequencing at the NCI Center for Cancer Research Genomics Core. The phRL-TK renilla luciferase construct was used as a control to normalize for transfection efficiency. Primary hepatocytes were seeded into 12-well plates (4 × 10 4 cells/well). PPRE reporter constructs were co-transfected into hepatocytes with PPARA and RXR expression. vectors. PPRE-luc plasmid containing an Acox1 PPRE site repeat was used as a positive control (Kliewer et al., 1992) . Empty pGL4.11 plasmid was used as negative controls. Plasmids were transfected using Lipofectamine 3000 Reagent (Thermo-Fisher Scientific). Luciferase activities were measured and plotted relative to lysate protein concentrations using the Promega Dual Luciferase Reporter (Promega) assays according to the manufacturer's protocol. Measurements were taken on a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA, USA).