Hepg2

The human cell lines HEK293, HEK293T, the human glioblastoma T98G, the human lung cancer H1299 and human ovarian cancer ES-2 were obtained from the American Type Culture Collection (ATCC; Rockville, MD). The human HCC cell lines, HepG2, PLC/PRF/5 and HUH7 were purchased from the JCRB cell bank (National Institute of biomedical Innovation) (Osaka Japan). The glioblastoma cell line U251 was purchased from the NCI Repository.

Human HCC cell lines HLE, JHH5, HuH‐7, and HepG2 were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Li‐7 cells were purchased from the RIKEN BioResource Center (Ibaraki, Japan). Hep3B cells were purchased from American Type Culture Collection. HLE, HuH‐7, HepG2, and Li‐7 cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS). Hep3B cells were cultured in Eagle's Minimum Essential Medium supplemented with 10% FBS and JHH5 cells were cultured in Williams's medium with 10% FBS.

The human hepatocellular cell lines Huh7 and HepG2 (Japanese Collection of Research Bioresources, Osaka, Japan) were grown and maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air, and the culture medium was changed three times per week.

HepG2 and Huh-7 cell lines were purchased from JCRB Cell Bank. Huh-7 was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai tesque), and HepG2 was maintained in DMEM with high glucose (Nacalai tesque). All culture media contained 10% fetal bovine serum (FBS; Gibco) and 50 units/50 μg/mL penicillin-streptomycin (Nacalai tesque).

HuH1, HuH7, HLE, HLF, Hep3B, HEP-G2, SK-Hep-1, and PLC/PRL/5 human liver cancer cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB; Osaka, Japan) or American Type Culture Collection (ATCC; Manassas, VA). Cells were routinely cultured in DMEM supplemented with 10% FBS. Two fresh HCC specimens (HCC1 and HCC2) were obtained and were used for xenotransplantation and to prepare single-cell suspensions for analysis. Primary HCC tissues were dissected and digested in 1 mg/mL type 4 collagenase (Sigma-Aldrich Japan K.K., Tokyo, Japan) solution at 37 °C for 15–30 min. Contaminated red blood cells were lysed with ammonium chloride solution (STEMCELL Technologies, Vancouver, BC, Canada) on ice for 5 min.

The human HCC cell lines, HepG2 and PLC/PRF/5, were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan) and American type culture collection (ATCC), respectively. HepG2 and PLC/PRF/5 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 1% penicillin/streptomycin solution and 10% fetal bovine serum (Gibco BRL., Grand Island, NY, USA) in a humidified 5% CO₂ incubator at 37 °C. The target sequence of HOXB13-siRNA was as follows: 5-(GGUCAUGGUUUGUUGAGCA)-3 (Ambion, Cat. 4392421). Lipofectamine (Invitrogen, Waltham, MA, USA) and HOXB13-siRNA were mixed in Opti-MEM (Thermo Scientific, Rockford, IL, USA). After 48 h of HOXB13-siRNA transfection, RNA was extracted. Total cellular RNA was extracted using the QIAzol lysis reagent (Qiagen, Redwood City, CA, USA) according to the manufacturer’s protocol. A NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the quantity and quality of isolated total cellular RNA.