Aml12 mouse hepatocytes

AML12 mouse hepatocytes (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM/F12 with 10% FBS supplemented with 1% Insulin-Transferrin-Selenium (ITS) (51500056; Invitrogen, Waltham, MA, USA), 40 ng/mL dexamethasone, and 1% penicillin streptomycin mixture. The plRES2-EGFP-NC and plRES2-EGFP-Hamp plasmids were transfected into AML12 cells using the EndoFectin™ Max transfection reagent (GeneCopoeia, Rockville, MD, USA) following the manufacturer’s instructions. Briefly, AML12 cells (5×10⁴ per well) were seeded into a 12-well plate. After 24 h, the cells were transfected with either 2 or 4 µg of the plasmids using the EndoFectin™ Max transfection reagent.

Livers from control or CCl₄-treated male wild-type Swiss Webster mice (6–8 weeks) were used for isolation of primary HSC or hepatocytes. Livers were perfused in situ and then subjected to either collagenase digestion for isolation of hepatocytes [16] or pronase/collagenase digestion and buoyant-density centrifugation for isolation of HSC [16,22,23]. As we have previously described [19], quiescent HSC isolated from control animals contained lipid droplets and were positive for desmin or Twist1, but not for CCN2, αSMA or collagen α(I) whereas activated HSC isolated from animals treated with CCl₄ were positive for desmin, CCN2, αSMA or collagen α(I), but not for Twist1 (data not shown). Hepatocytes were maintained in complete William E medium (Gibco, Billings, MT) and used within the first 3 days of primary culture while HSC were split 1:3 in DMEM/F12/10% FBS medium (Gibco) every 5 days and used at up to passage 4 (P4). LX-2 human HSC (a kind gift from Dr Scott Friedman, Icahn School of Medicine at Mount Sinai, New York, NY) were cultured in DMEM/10% FBS as described [19,22]. AML12 mouse hepatocytes (American Type Culture Collection, Manassas, VA) were cultured in DMEM/F12/10% FBS supplemented with insulin, transferrin, selenium and dexamethasone [19].