Snu 387

Human (HepG2, Hep3B, Huh7, SNU-387, and SNU-475; ATCC, Manassas VA), murine (Hepa1-6; ATCC), and Oncopig HCC cell lines were maintained in DMEM (or DMEM/F12 for HepG2) supplemented with 10% FBS and 1% Penicillin-Streptomycin.

Doxycycline hyclate (DOX, Sigma); ICG-001 (Selleckchem); fetal bovine serum, DMEM and DMEM/F12 medium (Gibco); fibroblast growth factor (FGF) and epidermal growth factor (EGF) (PeproTech); Trizol reagent (TAKARA); protease and phosphatase inhibitor cocktail (Roche); Lipofectamine 3000 and B27 (Invitrogen); dual-specific luciferase assay kit (Promega); Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies); Western blotting substrate (Millipore); Cell Signaling Senescence β-Galactosidase Staining Kit (CST); silver staining Rapid silver staining kit's (Beyotime); BALB/c nude mice (Beijing Vital River Laboratory Animal Technology); antibody against GATA4, Lamin B1, P21, FLAG, P15 and c-MYC (Abcam); HA and β-actin (Sigma); β-catenin, LEF1, TCF1, P14, P27, P53, p14/ARF, Caspase-9 and Caspase-3 (CST), P16/Ink4a (Epitomics); Cytokeratin (AE1/AE3) antibody (Kit-0009, MXB Biotechnologies) were purchased from the indicated manufacturers. SNU-387, SNU-449, PLC, NeHepLxHT, SK-Hep1, HepG2, HUH7 and HEK293 cells were obtained from ATCC.

SNU387, SNU423, SNU475, Huh7, PLC/PRF/5 (HCC), HepG2 (Hepatoblastoma) and SKHep1 (adenocarcinoma of liver) were purchased from ATCC. Hep40 cells (HCC) were kindly provided by Prof. M. Ozturk (IBG, Izmir, Turkey). Cells were propagated in DMEM (PAA) supplemented with 10% FCS, Penicillin/Streptomycin (50 U/ml) and 2 mM L-Glutamine in a humidified, 5% CO₂ incubator. The authenticity of Hepatoma cell lines were validated by STR analysis (Eurofins, Germany) and sequencing of p53 cDNA as they contain different p53 mutations^{12 (link)}. MycoAlert Mycoplasma Detection Kit (Lonza) has been used routinely to check mycoplasma contamination. Full-length human ZEB1 and ZEB2 cDNA were cloned into pCDNA4 plasmid with N-terminal HA-tag and sequence verified. E-Cadherin promoter luciferase reporter was used as described before^{11 (link)}. Transfections were performed using Lipofectamine LTX reagent (Invitrogen). Where necessary, cells were treated with TGFβ (R&D systems), UCN-01 and Midostaurin (Enzo Lifesciences), Oxaliplatin (Hospira, UK) and Sorafenib (Bayer, UK). All other chemicals were obtained from Sigma. Small hairpin RNA constructs (control and validated PKCα targeting, TRC no: TRCN0000195322-PKC-sh-1 and TRCN0000001693-PKC-sh-2) were purchased from Sigma.

Human HCC cell lines HEP3B and SNU387 were purchased from ATCC. Human HCC cell lines Huh7 and MHCC-97H were purchased from the National Collection of Authenticated Cell Cultures (https://www.cellbank.org.cn/). HEP3B cells were cultured in Minimum Essential Media (MEM, Gibco, 11095080) Supplementaryemented with 1% Non-Essential Amino Acids (NEAA, Sigma Aldrich, M7145) and 10% fetal bovine serum (FBS, Gibco, 12662029). Huh7, MHCC-97H, and SNU387 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 11995065) with 10% fetal bovine serum (FBS, Gibco, 12662029). All cells were cultured in a humidified incubator containing 5% CO₂ at 37 °C.

The HCC cell lines SNU398, SNU387, Huh7, HepG2, and PLC/PRF/5 were obtained from ATCC. Cells were grown in Roswell Park Memorial Institute (RPMI) medium or Dulbecco’s Modified Eagle’s Medium (DMEM); supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin; and maintained at 37°C with a 5% CO2 atmosphere. Regorafenib (#CS-1205) and sorafenib (#CS-0164) were purchased from ChemScene. Lenvatinib (#19375) and cabozantinib (#18464) were purchased from Cayman Chemical. Trametinib (#S-2673) was purchased from Selleck Chemicals, and buparlisib (#HY-70063) was from MedChem Express. Recombinant human IFNγ (#300-02) was obtained from PeproTech. Cell viability was quantified with Cell Count Reagent SF (nacalai tesque). Absorbance at 450 nm was measured on a micro- plate reader. For quantification of cytotoxicity, we used LDH Cytotoxicity Assay Kit (nacalai tesque). The absorbance value at 490 nm was measured.

The human hepatic normal cell line HL7702 and liver cancer cell lines QGY-7703 and SMMC7721 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China ), and cultured in high glucose DMEM (HyClone, Logan, USA) containing 10% FBS. The liver cancer cell line HepG2 was purchased from the National Collection of Authenticated Cell Cultures (Beijing, China) and cultured in RPMI 1640 (HyClone) supplemented with 10% FBS (HyClone). Human liver cancer cell lines, including SNU387, Huh7, and HCCLM3 cells, were obtained from ATCC (Manassas, USA) and cultured in high glucose DMEM (HyClone) containing 10% FBS. All cell lines were maintained at 37°C with 5% CO
₂ in a humid atmosphere. Cell lines were tested for mycoplasma contamination using polymerase chain reaction.