Fa2n 4

The immortalized hepatocyte cell line Fa2N-4 (Xenotech, Kansas City, KS, USA) was maintained in BEGM Bullet kit medium with 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Fa2N-4 cells were plated in 6-well plates to achieve ~80% confluence next day. Cells were collected at 24 h after treatment with vehicle or baicalin (0–100 μM) in the presence or absence of 3ʹ,4ʹ-DMF (2 μM).
The human hepatocellular carcinoma cell line HepG2 (ATCC, Manassas, VA, USA) was maintained in DMEM containing high glucose, 10% heat-inactivated FBS, and 1% antibiotic-antimycotic solution (Invitrogen) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The cells were treated with baicalin (1, 10, or 50 μM) for 48 h after pGL4.10-X1/2 plasmid transfection. BNF (50 μM) was used as a positive control.

The Hep3B, Huh7, PLC/PRF/5, and SNU449 HCC cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). Huh7.5 cells were kindly provided by Dr. Marc Windisch (Institut Pasteur Korea, Gyeonggi-do, Korea), and Huh6 cells were obtained from Cell Bank Australia (Westmead, NSW, Australia). Human immortalized hepatocytes (Fa2N-4) were obtained from XenoTech (Lenexa, KS, USA). All cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂. Dulbecco’s Modified Eagle’s Medium (Welgene, Daegu, Korea) was used to cultivate the Hep3B, Huh7.5, and Huh6 HCC cell lines, and Roswell Park Memorial Institute 1640 (RPMI1640) medium was used to cultivate the Huh7, PLC/PRF/5, and SNU449 HCC cell lines. DMEM and RPMI1640 media were supplemented with heat inactivated 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1× penicillin-streptomycin (P/S; Gibco). Fa2N-4 cells were plated on collagen-coated plates (BD Biosciences, San Jose, CA, USA) in serum-containing plating medium (plating media; XenoTech), which was replaced with supporting culture medium (XenoTech) after cell attachment (approximately 3–6 h).

The HCC cell lines ;SNU449, SNU475, SNU398, SNU898, Huh7, HepG2, Hep3B and PLC/PRF/5 were purchased from the Korean Cell Line Bank. Huh6 cells were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Germany). All HCC cells were maintained in RPMI (Welgene, Korea) or Dulbecco’s Modified Eagle Medium (DMEM; Welgene, Korea) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (p/s; Gibco, Grand Island, NY, USA). Fa2N-4, a human immortalized hepatocyte cell line, was obtained from Xenotech (Lenexa, KS, USA) and first cultured in serum-containing plating medium (K4000; Xenotech). Miha. Cells were kindly provided by Dr. Kim. K. M. from ASAN medical center. To produce tumor spheroids, HCC cells seeded at a density of 6 × 10³ cells/well in 96-well round-bottom ULA microplates (Corning Life Sciences, Corning, NY, USA). All cells were maintained in a 5% CO₂ humidified incubator at 37 °C.