L wnt3a

The human CRC cell lines HT-29 (ATCC, HTB-38), HCT-116 (ATCC, CCL-247), Caco-2 (ATCC, HBT-37), SW480 (ATCC, CCL-228), LoVo (ATCC, CCL-229), L-Wnt3a (ATCC, CRL-2647) and the parental line L-cells (ATCC, CRL-2648) were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM – Invitrogen Corporation, Carlsbad, CA, USA). SW480 cell line was grown in DMEM/ Ham′s Nutrient Mixture F12 and LoVo cell line was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium. All mediums were supplemented with 10% fetal bovine serum (FBS), penicillin G (100 mg/L), and streptomycin (60 mg/L) (Invitrogen) and cultivated at 37°C in a humidified atmosphere containing 5% CO₂. Cells were passaged weekly by using a solution of 0.05% trypsin/0.02% EDTA in PBS. A table with the most noticeable mutations already described for each cell line is provided as Supplementary Material 5.
Prior to pharmacological treatments, cells were maintained overnight in DMEM with 1% FBS and then treated with Wnt3a (100 ng/mL), 50% conditioned medium of Wnt3a, or IGF1 (100 ng/mL) for different times. For experiments with the PI3K inhibitor, cells were pre-treated with 15 µg/mL of LY294002 1 h before the treatments described above.

Luciferase assays were conducted as previously described [26 (link)]. Cells were seeded in quadruplicate in a 24-well plate (5 × 10⁴ cells per well) and transfected with Lipofectamine 2000. Each reaction contained 100 ng of the luciferase reporter plasmid and 2 ng pLRL-SV40 Renilla, serving as a transfection control. Total concentration of DNA was adjusted to 500 ng per reaction using pBluescript II SK (-). Where indicated, 50 ng of pcDNA3.1-β-catenin S45F and 50 ng of pME18-Lef1 were added to the transfection reaction. Transfection media were replaced after 6 h. For Dox-induced TCF7L1 or active Wnt signaling experiments, transfection media were replaced with Dox-treated media and/or Wnt3a-conditioned media (L-Wnt3a, ATCC, CRL-2647, Gathersburg, MD, USA), respectively. After 24 h, cells were lysed in passive lysis buffer (Biotium, #99821, Fremont, CA, USA) and luciferase levels were measured using the dual luciferase single tube assay kit (Biotium, #30081) on a Glomax 20/20 single chamber luminometer (Promega, Madison, WI, USA).

Murine pancreatic cancer cells (mPDAC) were cultured as previously described (Gilles et al., 2016) [2 (link)]. MIA PaCa-2, PAnc-1, and L-Wnt-3A cells were purchased from ATCC (Manassas, VA, USA) and cultured according to the manufacturer’s instructions. Wnt3a conditioned medium (Wnt3a-CM) was produced and recovered as described by Shibamoto et al. [30 (link)], and 50% Wnt3a-CM was added to basal cell medium to activate the Wnt/β-catenin pathway.

All cells were obtained from ATCC. HCT116 cells (ATCC CCL-247) were maintained in McCoy’s 5A Modified Medium; 293T cells (CRL-3216) were maintained in Dulbecco’s Modified Eagle’s Medium (“DMEM”); L-Wnt3a (CRL-2647) and L-Control (CCL-1.3) were used to make Wnt3a-conditioned medium, per ATCC protocol. All media was supplemented with 10% Tet System-Approved FBS (Clontech) and 1x penicillin-streptomycin. Cells were dissociated using TrypLE Express (Life Technologies) and were split at a ratio between 1:4 and 1:8 every 3–4 days.

Human fetal astrocytes (HFA), isolated at approximately 20 weeks of gestation, were purchased from Lonza (Lonza Biologics, Portsmouth, NH). U138MG cells were obtained from ATCC (ATCC HTB-16, Manassas VA) and cultured as described in ([12] (link)). Human Progenitor-Derived Astrocytes (PDA) were generated from neural progenitor cells, as previously described ([13] (link)). Briefly, progenitor cells were provided by Dr. Eugene Major (National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD (NIH) and seeded on poly-D-lysine- coated T-75 tissue culture flasks at 2×10⁶ cells/flask. Cells were maintained in progenitor medium consisting of neurobasal media (Life Technologies Invitrogen, Carlsbad, CA) supplemented with 0.5% bovine albumin (Sigma, St. Louis, MO), neurosurvival factor (Lonza), N2 components (Life Technologies Invitrogen), 25 ng/ml fibroblast growth factor, 20 ng/ml epidermal growth factor (R&D Systems, Minneapolis MN), 50 μg/ml gentamicin (Lonza) and 2 mM L-glutamine (Life Technologies Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM (Life Technologies Invitrogen) supplemented with 10% heat-inactivated FBS (Sigma), 2 mM L-glutamine, and 50 μg/ml gentamicin. L-Wnt3a (ATCC CRL-2647) were cultured in complete DMEM with 0.4 mg/ml G-418. Media was supplemented every three days.