Leibovitz s l 15 medium

Hepatic differentiation from human iPSCs was performed as described previously ^{(5) (link)} with some modifications. Confluent human iPSCs were passaged at a split ratio of 1:3 on iMatrix-511 with Essential 8^® medium and cultured for 2 d. To initiate differentiation, the cells were subsequently treated with RPMI-1640 plus 2% B27 Minus Insulin (RPMI-B27), containing 3-6 μM CHIR-99021 (MCE, NJ, USA #HY-10182) and 1% GlutaMAX^® (Invitrogen) for 24 h, followed by treatment with RPMI-B27 alone for 24 h. To initiate differentiation into hepatic progenitors, the cells were treated with RPMI-B27 containing 1% GlutaMAX^®, and 1% dimethyl sulfoxide (DMSO; Wako) for 5 d. The cells were detached using TrypLE Select (Invitrogen) and re-seeded on culture dishes coated with Matrigel (Growth Factor Reduced; Corning, Inc.) in hepatocyte maturation medium: Leibovitz’s L-15 medium (Wako) containing 8.3% tryptose phosphate broth, 10 μM hydrocortisone 21-hemisuccinate, 50 μg/mL sodium-L-ascorbate, 100 nM dexamethasone (DEX) (all from Sigma-Aldrich), 0.58% insulin-transferrin-selenium (ITS), 2 mM GlutaMAX^® (all from Invitrogen), 8.3% fetal bovine serum (Biowest), and 100 nM Dihexa (TRC, Ontario, Canada #H293745). This culture was continued for 20 d to obtain definitively differentiated hepatocyte-like cells. During the culture, the medium was changed every 2 d.

PH5CH8 immortalized human primary hepatocytes, A549 human lung carcinoma cells, 293T and 293FT human embryonic kidney cells, and HepG2 and Huh-7.5 human hepatoma cells were mycoplasma-free and cultured in Dulbecco's modified Eagle's medium (DMEM), High Glucose supplemented with 10% fetal bovine serum (FBS), 1 × GlutaMAX-I and 1 × MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific) at 37°C in a 5% CO₂ atmosphere. AML12 immortalized mouse hepatocytes were grown in DMEM/F-12 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 1 × Insulin-Transferrin-Selenium (Gibco), 40 ng/ml dexamethasone and 2 mM GlutaMAX-I. Primary human hepatocytes (PXB cells) were purchased from PhoenixBio Co. and maintained in Leibovitz's L-15 medium (Wako) supplemented with 26 mM NaHCO₃, 100 IU/ml insulin, 1 μM hydrocortisone and 10% FBS (Gibco).

Gefitinib was purchased from Tokyo Chemical Industry. Cycloheximide was purchased from Wako. phorbol 12-myristate 13-acetate was purchased from Sigma. Peptides were purchased from GenePharma. Amino acid sequences of peptides were described in Table 1. SW620 and MDA-MB-453 cells were cultured in Leibovitz's L-15 Medium (Wako) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) at 37 °C in a humidified atmosphere without CO₂. Other cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% FBS and 0.05 mM 2-mercaptoethanol (Nacalai Tesque) at 37 °C in a humidified atmosphere with 5% CO₂.

Human breast cancer cell line MDA-MB-231 was obtained from ATCC (Manassas, VA, USA) and MCF-7 cell line was obtained from RIKEN Cell Bank (Tsukuba, Japan). Cell line authentication was performed using Gene Print 10 System (Promega, Madison, WI, USA). The short tandem repeat profiles were completely matched with the registered database of ATCC in both MDA-MB-231 and MCF-7 cells. MCF-7 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C with 5% CO₂ and MDA-MB 231 cell line was cultured in Leibovitz’s L15 medium without CO₂. DMEM was purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Leibovitz’s L15 medium was purchased from Wako Pure Chemical Industries (Osaka, Japan). They were used with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Wako Pure Chemical Industries).