Small interfering rnas sirnas

Human hepatoma cells (HepaRG, Thermo Fisher, San Jose, CA, USA) were maintained in DMEM supplemented with 10% FBS (Hyclone, Logan, UT, USA). Primary murine hepatocytes were isolated as previously described^{24 ,25} and seeded at 2 × 10⁵ cells/well for 12-well culture dishes, or 4 × 10⁵ cells/well for 6-well culture dishes, or 4 × 10⁶ cells/well for p100 culture dishes. Cell viability was examined at the time of seeding by trypan blue staining; typical isolation yielded >95% viability. EGR1 promoter-luciferase construct was generated by amplifying genomic DNA spanning the proximal promoter and the first exon of EGR1 gene (-900/+50) and ligating into a pGL3-basic vector (Promega, Madison, WI, USA). Truncation mutants were made using a QuikChange kit (Thermo Fisher Scientific, Waltham, MA, USA) and verified by direct sequencing. Small interfering RNAs (siRNAs) were purchased from Dharmacon (Lafayette, CO, USA). Transient transfections were performed with Lipofectamine 2000 (Thermo Fisher Scientific). Luciferase activities were assayed 24–48 h after transfection using a luciferase reporter assay system (Promega) as previously described.²⁶