Dulbecco modified eagle medium (dmem)

Diploid or tetraploid blastocysts 4.5 dpc were transferred to a gelatin (Millipore)-coated cell culture dish with mitomycin-C-treated mouse embryonic fibroblast (MEF) feeder cells and cultured in stem cell medium comprising DMEM (Gibco) with 20% FBS (Gibco), 1% NEAA (Gibco), 1% GlutaMAX-L (Gibco) and 1% penicillin/streptomycin (Gibco); 0.1 mM b-mercaptoethanol; and 1,000 units/ml ESGRO leukemia inhibitory factor (LIF; Millipore). After 6–7 days, the colonies were digested with TrypLE (Invitrogen), transferred to a new gelatin-coated cell culture dish with mitomycin C-treated MEFs, and cultured in fresh medium; these cells were designated as P1. Then, the ESCs from P2 were cultured in stem cell medium comprising DMEM with 15% FBS, 1% NEAA, 1% GlutaMAX-L, 1% penicillin/streptomycin, 0.1 mM b-mercaptoethanol, 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck) and 1,000 units/ml LIF.

Clonally expanded murine CD133 positive (TICs) cells (kindly provided by Dr. Bart Rountree) originally described by Rountree et al. [11 (link)] was cultured on standard tissue culture plates (BD Biosciences) in DMEM (Invitrogen) containing 10% Fetal Bovine Serum (FBS, Invitrogen), 100 units/100 μg/ml Penicillin/Streptomycin (Gibco, Carlsbad, CA), 1 μg/ml Insulin (Sigma-Aldrich, St. Louis, MO), 10^− 7 M dexamethasone (Sigma-Aldrich), and 10 mM nicotinamide (Sigma-Aldrich) in a cell culture incubator maintained at 37 °C and 5% CO₂. Human hepatocellular carcinoma cell line HepG2 and cholangiocarcinoma cell line MzCha-1 (kindly provided by Dr. Shelly Lu, Cedars Sinai Medical Center) were grown as described previously [22 (link)]. Cells were trypsinized with 0.05% Trypsin-EDTA (Gibco, Carlsbard, CA) and passaged every 3 days. For serum starvation, cells were washed in sterile phosphate buffered saline (PBS) twice and media was changed to DMEM containing dexamethasone and nicotinamide minus serum and Insulin for 16 h. For CBP loss-of-function experiments, we utilized ICG001 (Selleckchem, Houston, TX), a small molecule inhibitor that specifically disrupts beta-catenin interaction with CBP at an established dose of 10 μM concentration as described previously [19 (link), 21 (link), 23 (link)].

Primary human AoSMCs were purchased from Lonza, grown for 5–8 passages in SMGM2 culture medium supplemented with 5% FBS, and maintained at 37 °C in the presence of 5% CO₂. Briefly, 1.2 × 10⁵ cells were plated in a 35-mm dish and incubated for 24 h with DMEM (Euroclone) containing 0.2% FBS to induce starvation. After 24 h, DMEM was replaced with complete medium, and cells were treated for 24 h with 1 μm SRT1720 (Selleck Chemicals), 100 μm RESV (Selleck Chemicals), and/or with 0.1 μg/ml TNFα (Abnova) to induce the inflammatory responses. To inhibit p65 nuclear translocation, cells were treated with 10 μm ammonium pyrrolidinedithiocarbamate (PDTC, Sigma) for 24 h.

The mouse macrophage cell lines J774A.1 (ATCC TIB-67) and RAW264.7 (ATCC TIB-71) were maintained and cultured in DMEM (Hyclone, USA) supplemented with 5% fetal bovine serum (FBS) (Thermo Fisher, USA) (34 (link)). For infection experiments, macrophages were infected with a single cell suspension of M. bovis BCG Pasteur strain 1173P2, at a multiplicity of infection (MOI) of 1:10 for 3 h. Subsequently, the extracellular bacteria were removed by washing twice with 1× PBS and cells were overlaid with DMEM medium containing MK-2206 (Selleckchem, USA).