Hep g2 cells

HEK293 cells (American Type Culture Collection [ATCC], Manassas, VA; cat. no. CRL-1573) and Hep G2 cells (ATCC; cat. no. HB-8065) were maintained in Dulbecco’s modified Eagle’s medium and Roswell Park Memorial Institute, respectively, supplemented with glucose (4.5 g/L), 4 mM glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). Hep G2 cells were transfected with plasmids using Neon Transfection (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Briefly, 1.5 × 10⁵ cells were resuspended to 10 μL of R buffer in Neon Transfection kit. Subsequently, 1230 V/20 ms/3 pulses Neon system is used to electroporate the cells. Two days after transfection, the cells were harvested, and genomic DNA was extracted using a FavorPrep blood/cultured cell genomic DNA extraction mini kit (FAVORGEN). For transfection of HEK293 cells, 5 × 10⁴ cells were seeded the day before transfection in 24-well plates. The next day, Total 1 μg plasmids encoding human optimized F9 donor and CjCas9 was diluted to 200 μL Opti-MEM and mixed with diluted lipofectamine 2000 (1:2 ratio) and then incubated for 20 min and added to cells. Genomic DNA was isolated after 4 days of transfection.

OP9 mouse stromal cell and HepG2 cell were obtained from the American Type Culture Collection (ATCC, USA). HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Life Technologies). Palmitic acid (PA) and oleic acid (OA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HepG2 cells were incubated with 300 μM oleic acid (OA) plus 300 μM Palmitic acid (PA) for 48 h, as described previously. Subsequently, the medium was replaced with a normal fresh medium containing RA at final concentration of 10 μM and incubation was continued for an additional 48 h. OP9 cells were maintained in MEM-α containing L-Glutamine, with 20% FBS (Secure foetal bovine serum; Gibco, Australia), 100 U/mL penicillin, and 100 μg/mL streptomycin [21 (link)]. For adipocyte differentiation, 1 μM rosiglitazone was added to the culture medium for differentiation at 37°C in 5% CO₂. RA was then added to the medium at 10 μM for 5 days. Three wells of OP9 cells were used for each experiment. After treatment for 5 days, the cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, and then stained with oil red O (Sigma, USA). The absorbance was measured at 510 nm.