Cm4000

The hepatocytes were prepared by standard procedures at Yale Liver Center, spun down at 50g, plated on collagen-coated dishes at a density of 10⁵/cm², and maintained in William’s E media plus 10% FBS plus 1× supplement (Thermo Fisher Scientific, CM4000) plus 1 μM dexamethasone in a 5% CO₂ incubator. For in vitro transduction experiments, the titrated AAV8 viral particles were transduced at an MOI of 60 in William’s E media plus 1 nM insulin plus 1 μM dexamethasone for 24 hours. Seventy-two hours after transduction, cells were fixed in 10% formalin for Oil Red O staining.
Seahorse experiments for determining β-oxidation of fatty acids. The β-oxidation assay was carried out with the Seahorse XF Cell Mito Stress Test Kit per the manufacturer’s instructions (Agilent, part 02720-100) in the 96-well format with hepatocyte density of 2 × 10³ cells per 100 μL. The Mito Stress Test (oligomycin 2.5 μg/mL, FCCP 0.8 μg/mL, 2 μM rotenone) was performed under the following 4 conditions: BSA, BSA + 400 μM etomoxir, BSA-palmitate, and BSA-palmitate + 400 μM etomoxir. For virus transduction experiments, the cells were incubated in AAV8 in William’s E media plus 1 nM insulin plus 1 μM dexamethasone, for 24 hours before the assay.

Primary human hepatocytes are thawed in Cryopreserved Hepatocyte Recovery Medium (CHRM, CM7000, ThermoFisher) and plated using Primary Hepatocyte Thawing/Plating supplements (CM3000, ThermoFisher), with a 1:40 dilution of Matrigel (354277, Corning) and at least 3% fetal bovine serum (FBS). 24 h post-plating, media is changed and Primary Hepatocyte Maintenence Supplements (CM4000, ThermoFisher), plus 1% FBS are added. All supplements are added to Williams E Media, with no phenol red (A1217601, ThermoFisher).

Primary human hepatocytes (PHH) were purchased from Thermofisher scientific (HMCPMS; Thermofisher Scientific, MA, USA), and were cultured and maintained according to the supplemented protocol. In brief, the hepatocytes were thawed in Cryopreserved hepatocytes recovery medium (CM7000; Thermofisher Scientific), centrifuged at 100 x g for 10 min, and seeded at 2 × 10⁶ cells/well on a collagen-coated plate in cryopreserved hepatocyte plating medium (CM9000; Thermofisher Scientific). After incubating the plate at 37 °C for 6 h, the culture medium was replaced by William’s medium (Gibco) supplemented with hepatocytes maintenance supplement pack (CM4000; Thermofisher Scientific).

Cryopreserved PHH from single donors (BD Gentest, Tewksbury, MA; Lonza, Walkersville, MD; and Thermo Fisher Scientific) were used for all in vitro experiments. PHH were thawed according to the supplier’s instructions using Cryopreserved Hepatocyte Recovery Medium (CM7000; Thermo Fisher Scientific), resuspended in Williams’ Medium E (MilliporeSigma, Burlington, MA) with plating supplements (CM3000; Thermo Fisher Scientific), and plated at a density of 50,000 cells/cm². One day after seeding, PHH were serum starved overnight in serum-free Williams’ Medium E with maintenance supplements (CM4000; Thermo Fisher Scientific). Cells were then incubated for 48 h in DMEM supplemented with 0.5% BSA (free fatty acid free; MilliporeSigma) and 1% penicillin–streptomycin with or without the addition of 400 μM sodium oleate (68 (link), 69 (link)).
For albumin measurements, media were collected at the end of 48 h of lipid loading, and albumin concentration was measured with a commercially available human albumin ELISA kit (Invitrogen, Waltham, MA).
After 48-h lipid loading, media were changed to DMEM with 1% penicillin–streptomycin with or without the addition of cytoskeletal drugs: 5 µM blebbistatin (MilliporeSigma), 5 µM latrunculin (MilliporeSigma), or 10 µM nocodazole (Cayman Chemicals, Ann Arbor, MI). Cells were treated for 4 h before fixation.