Hepg2

Two different cell lines, HepG2 and NIH 3T3 (both from ATCC, Manassas, VA, United States) were cultured to ∼90% confluence after 21–25 h of growth under standard conditions (5% CO₂/95% O₂ at 37°C): Seeding densities for HepG2 and NIH 3T3 were 6.97 × 10⁴ and 6.67 × 10⁴ cells/cm², respectively. HepG2 cells were cultured in Eagle’s Minimal Essential Medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) of non-essential amino acid (NEAA) mixture, and 1 mM sodium pyruvate. NIH 3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) newborn calf serum (NCS) (Gibco, Paisley, United Kingdom). All culture media were supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). All cell media and supplements were obtained from Sigma-Aldrich (St. Louis, MO, United States), except serum (Gibco, Paisley, United Kingdom). The 96-well plates were from Corning Costar (Sigma-Aldrich, Brøndby, Denmark).

The normal liver cells (HepaRG) and HCC cell lines (SMMC-7721, HepG2.2.15, HepG2 and Huh7) were purchased from ATCC(Manassas, VA, USA). HCC cells were cultured in a RPMI 1640 medium containing 10% fetal bovine serum (FBS) and HepaRG cells in keratinocyte-SFM (Thermo Fisher, Jijie Technology, Wuhan, China), together with 100 U/mL penicillin(Baomanbio, Pudong, Shanghai, China) and 100 μg/mL streptomycin(Yita Bio, Pinggu, Haidian, Biejing, China) with 5% CO₂ at 37°C.

The immortalized normal liver epithelial cells, THLE-3, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured under the conditions stated by the manufacturer. The HCC cell lines: HepG2, Hep3B, MHCC97H, MHCC97L, BEL-7402, Huh7, SMMC-7721, PLC/PRF/5, and QGY-7703 were kindly donated by Prof. Qian Wang (Sun Yat-Sen University, Guangzhou, China), which HepG2, Hep3B and PLC/PRF/5 were purchased from ATCC, and MHCC97H, MHCC97L, BEL-7402, Huh7, SMMC-7721 and QGY-7703 were purchased from Shanghai Yansheng industrial Co. (Shanghai, China). The HCC cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL), at 37°C in a 5% CO₂ atmosphere in a humidified incubator.

Human hepatoma cell lines HepG2 and C3A, a subclone of HepG2 (ATCC HB-8065), were purchased from the ATCC and cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen). Lung cancer cell line A549 was obtained from the ATCC and maintained in DMEM supplemented with 10% FBS. GP2-293 and Lenti-X 293T cell lines were purchased from Clontech and cultured in DMEM supplemented with 10% FBS and 1 mM sodium pyruvate (Invitrogen). Flp-In TREx 293 cells were purchased from Invitrogen and maintained in DMEM supplemented with 10% FBS, 10 μg/ml blasticidin (Invitrogen), and 100 μg/ml zeocin (Invivogen) (72 (link)). Flp-In TREx 293-derived cell lines expressing LY6E, GILT, ADAP2, or IFITM3 were cultured in DMEM supplemented with 10% FBS, 5 μg/ml blasticidin, and 250 μg/ml hygromycin.

HepG2 and THP-1 cell lines (American Type Culture Collection, ATCC, Manassas, VA, USA) were authenticated and stored in accordance with the instructions provided by the supplier. HepG2 and THP-1 cells were cultured in Eagle’s Minimum Essential Medium (ATCC) and Roswell Park Memorial Institute (RPMI)-1640 medium (Lonza, Verviers, Belgium), respectively, in the presence of 10% fetal bovine serum (FBS, Life Technologies, Monza, Italy) and 1% penicillin-streptomycin in a humidified 5% CO₂ atmosphere at 37 °C. THP-1 monocytes were plated and differentiated into macrophages (M0) with phorbol 12-myristate 12-acetatate (PMA) 100 nM for 24 h and then within a medium without PMA for 1 day, as described by Gionfriddo et al. [27 (link)]. M0 macrophages were exposed to 0.1 and 0.2 mg/mL of BL and BS, dissolved in distilled water and dimethylsulfoxide (DMSO), respectively, prior to treatment for 24 h with Lipopolysaccharide (LPS) 10 ng/mL, which was able to polarize M0 into the inflammatory M1 macrophages.