We performed a high throughput screen of twelve chemotherapeutic drugs that are known ABCG2 substrates on the mouse G3 MB Myc1 neurosphere, as previously described (22 (link)). Briefly, 1000 cells per well were plated in 384-well plates. After 24 hr, half of the plates were treated with 10 µM of FTC (Alexis Biochemical, San Diego, CA, USA), 10 µM of Ko143 (Tocris Bioscience, Bristol, UK), or 10 µM of Tariquidar (Sigma-Aldrich, USA), incubated for 30 minutes, and all plates were treated with 28 nl of compound in a dose-response (final drug concentration 9.3 µM to 0.5 nM). After 72 hr, the cell number was determined in each well using CellTiter-Glo© (Promega). The luminescence signal was measured in an automated Envision plate reader (Perkin-Elmer). Luminescence data were normalized by log10-transformation prior to calculating the percentage of inhibition using the following equation: 100 × (negative control mean − compound value)/(negative control mean − positive control mean). Cells treated with 1% DMSO served as negative control with 0% inhibition. Cells treated with 35 µM cycloheximide were used as positive control with 100% inhibition.