Hepg2 cells

HepG2 cells were obtained from Merck KGaA (Darmstadt, Germany; ECACC certified) and cultured according to the corresponding manufacturer’s instructions. During experiments, cells were cultured in serum-free medium. Cells were treated with 200 μM bovine serum albumin-conjugated palmitate (Cayman chemical, Ann Arbor, MI) alone or in combination with 10 μM of HK4 (Taros Chemicals, Dortmund, Germany) up to 30 min prior to palmitate exposure for 7 h.

HepG2 cells (Merck; 85011430-1VL) were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% supplementation of foetal bovine serum in a 5% CO₂ incubator. Cells were cultured in 10 ml of media in T75 flasks. The cells were passaged by washing with 5 ml PBS (1X) followed by incubation in 2 ml of 0.25% Trypsin-EDTA at 37 °C for 5 min. Trypsinisation was inhibited by adding 4 ml DMEM. Cells were then collected via centrifugation at 500×g for 5 min before being counted.

Human Caucasian Hepatocyte Carcinoma (HepG2) cells (Sigma Aldrich, UK) were utilized to assess SGC epigenetic probe cell cytotoxicity in the application of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [52 (link)]. Briefly, cells were passaged at 70–80% confluence and seeded (20,000 cells/ 50 μL per well) in a 96-well, black-sided, clear-bottomed falcon plate (Fisher Scientific, UK) with the final plate column (8 wells) being treated as a blank (media only). Following a 24 h incubation (5% CO₂, 37°C, humidified), HepG2 cells were then treated with 50 μL of pre-warmed media (37°C) containing SGC EPs/EIs at 200 μM to 0.02 μM (final concentration 100 μM, 10 μM, 1 μM, 0.1 μM and 0.01 μM). Three positive (1% Triton X-100) and negative (1% DMSO) control wells per plate were additionally included.
After addition of compounds, each plate was then incubated for a further 20 h before application of MTT for assessment of overt compound cytotoxicity using the MTT assay [52 (link), 65 (link)]. The MTT assay was read using the POLARstar Omega (BMG LabTech, UK) plate reader at an absorbance of 570 nm. CC₅₀s were calculated in GraphPad Prism.

The HEK293 cell line (American Type Culture Collection CRL-1573) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate. HepG2 cells (Sigma-Aldrich 85011430) were cultured in DMEM hi-glucose, supplemented with 10% (v/v) FBS. IMR32 cells were cultured in Roswell Park Memorial Institute-1640 media supplemented with 10% (v/v) FBS, 1% (w/v) penicillin-streptomycin (P/S), and 2.5% (v/v) Hepes. The undifferentiated human induced pluripotent stem (hiPS) cells (HipSci 77650065) were grown in eight Flex Media and 1% (w/v) P/S solution. The human neuroectodermal progenitor (NEP) stem cells were differentiated from the undifferentiated hiPS cells, according to (24 (link)). Human hiPS-derived hepatocytes were differentiated from a human undifferentiated hiPS cell line (101B) according to (25 (link)). The HuH7 cell line was grown in DMEM, high glucose, pyruvate, supplemented with 10% (v/v) FBS, 1% (v/v) glutamax, and 1% (w/v) P/S. All cells were grown to approximately 80% confluency in a 5% CO₂ incubator at 37 °C. The cells were subsequently harvested using trypsin-EDTA (0.25% (v/v)) and stored in a −80 °C freezer.

HepG2 cells were purchased from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC). HepG2 cells were cultured in Minimal Essential Medium (MEM) with Earle’s balanced salt solution (EBSS). The cell culture medium was supplemented with 2 mM of L-glutamine, 1.5 g/L of sodium bicarbonate, 0.1 mM of non-essential amino acids (NEAA), 1.0 mM of sodium pyruvate, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO₂. Before treatment, cells were seeded at a density of 5 × 10⁵ onto 6-well plates for 24 h. To induce overloading of fatty acids, HepG2 cells at 70% confluence were exposed to OA (Sigma-Aldrich, St. Louis, MO, USA). OA/bovine serum albumin (BSA) complex was prepared as reported previously [6 (link)]. Stock solutions of 1 M of OA prepared in culture medium containing 10 μL/mL of BSA were conveniently diluted in the culture medium to obtain the desired final concentrations. The OA/BSA complex solution was sterile-filtered through a 0.22 μm pore membrane filter and stored at −20 °C.

The inhibition of hepatic glycogenolysis was monitored by the measurement of liver glycogen, which was done quantitatively by the anthrone reagent (Sigma) colorimetric method based on the published method20 (link). Isolated rat hepatocytes or HepG2 cells (Sigma) were treated with the test compound or DMSO solvent (final concentration, 0.10%), followed by 60-min incubation with 0.3 nM glucagon (GGN). Assays were terminated by centrifugation, and cells were digested with 30% KOH followed by glycogen determination. The IC₅₀ values were estimated by fitting the inhibition data to a dose-dependent curve using a logistic derivative equation.

All experiments were approved by the Swiss Ethics Committees on Research Involving Humans. HepG2 cells were obtained from Sigma-Aldrich (Buchs, Switzerland). The cells (passage number 7 and 8) were cultured in DMEM low glucose (5 mM) (ref. 21885108, Thermo Scientific-Life Technologies, Zug, Switzerland) supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin/streptamin, and kept in a humidified atmosphere (5% CO₂) at 37 °C. The medium was changed every 2 days until experiment.
Differentiated human iPSCs (iCell astrocytes) were obtained from Cellular Dynamics International (CDI, Madison, WI, USA). The cells were thawed according to the manufacturer’s instruction. iPSC-derived astrocytes were cultured in high glucose (25 mM), pyruvate (1 mM)- and glutamine (4 mM)-containing DMEM (Thermo Scientific-Life Technologies, ref. 41966029) supplemented with 10% fetal calf serum and N2 complement. Cells were kept in culture in a humidified atmosphere (5% CO₂) at 37 °C for 7 days with the medium changed at days 3 and 6.