Insulin

Human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai). HUVECs were kept in RPMI-1640 medium from Invitrogen in Gaithersburg, Maryland, which also contained 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (complete medium). Human intestinal epithelial cells (HIECs) were supplied by Guangzhou Jennio Biotech Co., Ltd. Both HIEC and HCT116 (ATCC #CCL-247) cell lines were cultured in DMEM (Dulbecco’s modified Eagle’s medium, Thermo Fisher Scientific) containing 10% FBS at 37 °C in a humidified environment consisting of 95% air and 5% CO₂. All cell lines were passaged for fewer than 6 months after being revived. Artesunate (S2265), 5-fluorouracil (S1209), and insulin (S6955) were supplied by Selleck. H₂O₂ (10011208) was purchased from Wokai Biotechnology (Shanghai, China). They were reconstituted in dimethyl sulfoxide (DMSO) and kept at 20 °C for each experiment. The ultimate 5-FU concentration employed was 10 mmol/L in all experiments.

Serial dilutions of compounds were made in appropriate solvents (DMSO or H₂O) and corresponding carrier controls were included in the assays. Serial dilutions were made for single soluble factor titrations; FGF4, sodium (ortho)vanadate (Sigma-Aldrich, S6508), PD0325901, PD98059 (Sigma, P215), activin A, TGFβ1, A83-01 (Tocris, 2939), Nodal (R&D systems, 1315-ND-025), SB431542 (Tocris, 1614), retinoic acid (Sigma-Aldrich, R2625), DL-epinephrine HCl (Sigma-Aldrich, E4642), 8Br-cAMP, SC144 (Tocris, 4963/10), IL6 (Peprotech, 216-16), IL11, Lif, BMP4 (Peprotech, 315-27), BMP7, LDN 193189 (Tocris, 6053), ML347 (Selleckchem, S7148), Noggin (Peprotech, 250-38), CHIR99021, IWP2 (Selleckchem, S7085) and XAV-939 (Selleckchem, S1180). Compounds with end concentrations that were used for XEn/Epi EpiC modulation: PI3K inhibitor ZSTK474 (Selleckchem, S1072) at 1 μM, insulin at 50 ng/ml, XAV-939 at 20 μM.

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)].