Tankyrase inhibitor xav 939

Single-cell suspensions were plated on a 60-mm ultra-low attachment tissue culture dish (Corning, Lowell, MA, USA) at a density of 1×10⁵/dish in Dulbecco’s modified Eagle’s medium/F12 containing 10 ng/ml FGF-2, 4 μg/ml heparin, 20 ng/ml epidermal growth factor, 5 μg/ml insulin, and 5 μg/ml hydrocortisone for 7 days. Inhibition of mammospheres was tested with ICG001 (Tocris Bioscience), which was added at 5 μM or 10 μM or with 20 μM of Tankyrase inhibitor XAV 939 (Tocris Bioscience) in the medium for 7 days. For secondary sphere formation, primary spheres were dissociated in 1:1 trypsin/Dulbecco’s modified Eagle’s medium at 37 °C, and mechanically dispersed by passing through a 23 gauge needle. Single cells were replated at 5×10³/dish, and incubated in 37 °C 5% CO2 for 7 days. At the end of the treatment, cells were transferred to a 35 mm MatTek dish and spheres as well as total cells (including mammospheres, single cells and clusters) per microscopic field over five fields were counted. Mammospheres were expressed as percentage using the average number of spheres per 100 cells.