DAOY cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were grown as described previously [38 (link)]. BSZ2 cells were grown as described in [40 (link)] and PANC-1 (ATCC) cells as described in [46 (link)].
Ptch−/− and Sufu−/− embryonic fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum (FBS), antibiotics and for Sufu−/−cells also with L-glutamine. During assays and treatments of confluent cells, FBS was reduced to 0.5%. For 3-dimensional (3D) cultures, 5 × 103 cells were seeded in 12-well plates as described previously [62 (link)]. 3D spheroid cultures were grown for 4-6 weeks at 37°C in a humidified atmosphere containing 5% CO2. Colony formation was documented on a stereomicroscope with Cell^D Image capture system (Olympus, Tokyo, Japan) and quantified using Colony Counter Software (Microtec Nition, Chiba, Japan). Smoothened agonist SAG (Axxora, Farmingdale NY, USA), harmine (Thermo Fisher Scientific, Waltham, MA, USA), vismodegib, cyclopamine (LC Laboratories, Woburn, MA, USA), GANT61 (Merck Chemicals Ltd., Darmstadt, Germany) and DYRKi were dissolved in dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA).
Ptch−/− and Sufu−/− embryonic fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum (FBS), antibiotics and for Sufu−/−cells also with L-glutamine. During assays and treatments of confluent cells, FBS was reduced to 0.5%. For 3-dimensional (3D) cultures, 5 × 103 cells were seeded in 12-well plates as described previously [62 (link)]. 3D spheroid cultures were grown for 4-6 weeks at 37°C in a humidified atmosphere containing 5% CO2. Colony formation was documented on a stereomicroscope with Cell^D Image capture system (Olympus, Tokyo, Japan) and quantified using Colony Counter Software (Microtec Nition, Chiba, Japan). Smoothened agonist SAG (Axxora, Farmingdale NY, USA), harmine (Thermo Fisher Scientific, Waltham, MA, USA), vismodegib, cyclopamine (LC Laboratories, Woburn, MA, USA), GANT61 (Merck Chemicals Ltd., Darmstadt, Germany) and DYRKi were dissolved in dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA).
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