Wnt c59

Cells were grown in Dulbecco’s minimal essential medium (Gibco) with penicillin/streptomycin and 10% fetal bovine serum in a humidified atmosphere of 10% CO₂ at 37 °C. Experiments were performed with HeLa M cells, a sub-line of HeLa. HCT116 cells were obtained from Dr. B. Vogelstein^{61 (link)}, HT-1080 fibroblast cells were from Dr. G.R Stark and wild type and gsk3−/− mouse embryo fibroblast (MEF) were obtained from Dr. Jim Woodgett^{62 (link)}. Cells were synchronized by treating with 2 mM thymidine (Sigma Aldrich) for 20–21 hours for HeLa cells and 24 hours for HCT116 cells, followed by release for 9–10 hours before adding respective drugs.
Taxol (100 nM or 1 μM) (Cayman Chemical), Epothilone (20 nM) (Sigma-Aldrich) or Nocodazole (2 μM) were added to arrest cells in mitosis, after thymidine release where mentioned. The proteasome inhibitor MG132 (Cayman chemical) was used at a concentration of 20 μM and kinase inhibitors were used at previously determined effective concentrations: 2.5 μM ZM447439 (AstraZeneca), 2 μM Reversine (Calbiochem), 2.8 μM LY204002 (Cayman Chemicals), 30 μM SB415286 (Cayman Chemicals), 400 nM WNTc59 (Cayman Chemicals), RO 318220 (Cayman Chemicals), 60 mM Lithium Chloride, 14 μM RO3306 (Cayman Chemicals) and 10 μM Chelerythrine Chloride (Cayman Chemicals). Finally, cell selection was accomplished using puromycin at 2 μg/μL (Invitrogen).

Human colorectal tumor tissue-derived ALI organoids were cultured as described previously [10 (link)]. The medium components were as follows: Advanced Dulbecco’s Modified Eagle’s Medium (DMEM) with 50% Wnt, Noggin and R-Spondin conditioned medium; GlutaMax; B-27 supplement; 100 μg/mL Primocin (Invitrogen, Carlsbad, CA, USA); 1 mM N-Acetyl-l-cysteine; 10 mM Nicotinamide (Sigma-Aldrich, St. Louis, MO, USA); 50 ng/mL mouse epidermal growth factor (EGF) (PeproTech, Inc., Rocky Hill, NJ, USA); 500 nM A83-01 (Adooq Bioscience, Irvine, CA, USA); and 3 μM SB202190; 10 μM Y-27632 (Cayman, Ann Arbor, MI, USA). Stem cell-related signal inhibitors were as follows: YO-01027 (Toronto Research Chemicals, Toronto, Canada); DAPT (Adooq Bioscience); WAV939; Wnt-C59; AY9944; and GANT61 (Cayman). Anti-cancer drugs were as follows: 5-FU (WAKO, Tokyo, Japan); Irinotecan (LC Laboratories, Woburn, MA, USA); and Oxaliplatin (Adooq Bioscience). Antibody sources were as follows: GLI-1 (Gene Tex, Irvine, CA, USA); CD44 (Bethyl Laboratories, Montgomery, TX, USA); c-Myc; and Nanog (Cell Signaling, Beverly, MA, USA). Secondary antibodies were as follows: Horseradish peroxidase (HRP) conjugated anti-rabbit IgG; HRP conjugated anti-goat IgG (Cayman); and HRP conjugated anti-mouse IgG (Millipore, Temecula, CA, USA).