Reporter assays

HepG2 cells (HB-8065) were obtained from ATCC (American Type Culture Collection). Cells were maintained according to ATCC culture conditions. Freshly thawed cells were passaged at least three times before performing assays. ~ 10,000 HepG2 cells/well in complete medium (Eagle's Minimum Essential Medium supplemented with 10% FBS and 1% P/S) were seeded in a 96-well plate and grown overnight. Each well was transfected with 0.25 μl lipofectamine 2000, 200 ng of the SMAD1/5/8 responsive reporter plasmid pGL3 [luc2P/BRE] pGL3.48 and 2 ng of the [Luc2P/hRluc/TK] vector (control luciferase reporter plasmid, Promega, E6921). Transfection medium was removed the following day, and replaced with assay medium containing BMP-4 (1 nM) ± Cer-Fc (0–600 nM). After adding assay medium, cells were incubated for 16 h and luciferase activity was detected with a homemade dual-glow luciferase assay [25] using a FluoStar Omega plate reader. Relative luciferase units (RLU) were calculated by dividing firefly luciferase units (fLU) with renilla luciferase units (rLU). IC₅₀ values were calculated using GraphPad.