Penicillin

HepG2 cells were purchased from ATCC (Menassas, VA, USA). After thawing, cells were plated in T-75 flasks and grown in Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS), L-glutamine 2 mM, sodium pyruvate 1 mM, non-essential amino acids 1X, penicillin 100 units/mL, and streptomycin 100 μg/mL (HyClone Laboratories, Logan, UT, USA).
When confluent, cells were trypsinated (0.05% trypsin/0.53 mM EDTA) and seeded at a ratio of 1:3. Subsequent passages were performed every 6 days. Immortalized human hepatic stellate cells (LX-2) were purchased from Millipore (Burlington, MA, USA). LX-2 cells were grown in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (HyClone Laboratories) containing 10% fetal bovine serum, penicillin 100 units/mL, and streptomycin 100 μg/mL in T-75 flasks. When confluent, LX-2 cells were sub-cultured like HepG2 cells. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. HepG2 and LX-2 cells were genotyped for the PNPLA3 rs738409 and resulted in homozygotes for the 148M allele variant.

The FHCC-98 human HCC cell line and the LX-2 human HSC line (5×10⁵ cells/100 mm culture dish) (Department of Cell Biology, Fourth Military Medical University, Xi’an, China), were cultured at 37°C in a humidified atmosphere, containing 5% CO₂ in DMEM, containing 5% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (HyClone Laboratories, Inc., Logan, UT, USA).
For co-culture, the FHCC-98 and LX-2 cells (5×10⁵ cells/6-well plate) were mixed at a ratio of 1:1 and cultured in DMEM, containing 5% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin.
The cells were grown until 70% confluent and were subsequently incubated with fresh DMEM. Following incubation for 24 h, the HCC-CM was collected and centrifuged at 600 × g for 10 min at room temperature to remove debris, filtered through a 0.2 mm filter (EMD Millipore, Billerica, MA, USA) and stored at −20°C until use.

Healthy peripheral blood mononuclear cells were separated over Ficoll-Hypaque gradients (MP Biomedicals, Aurora, OH, USA). PBMC were grown in RPMI 1640 (Invitrogen, San Diego, CA, USA), supplemented with 2 mM l-glutamine, 50 ng/mL, streptomycin, 50 units/mL penicillin, and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). All volunteers provided written informed consent in agreement with the Declaration of Helsinki to the use of their residual buffy coats for research aims with approval from the University Hospital of Salerno Review Board.
Human immortalized keratinocytes (HaCaT) were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with 2 mM l-glutamine, 50 ng/mL, streptomycin, 50 units/mL penicillin, and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT, USA). HaCaT cells were kindly provided by Giuseppe Monfrecola (Department of Experimental Dermatology, University of Naples, Naples, Italy).