Expression of the mitochondrially enriched EGFRvIII renders GBM cells more resistant to the EGFR inhibitor, Iressa. A, U87MG-EGFRvIII-MTS1 cells are significantly more resistant to a 48-hr Iressa treatment (0-100 uM) than U87MG-EGFRvIII cells. Survival rates were determined by the Celltiter Blue Cell Survival Assay. B,C, Clonogenic growth assay confirmed the results of Panel A. The assay was performed in 6-well cell culture plates as previously described . Seeded cells were treated with 1% DMSO or 12.5 μM Iressa in 1% DMSO for 24 hrs followed by medium replacement with fresh drug-free growth medium and culturing for 10-14 days. Colonies were stained with crystal violet blue solution for 1 hr, washed with water, dried and counted. Triplicate wells were used for each treatment and three independent experiments were performed to derive means and standard deviations. Student t-test was performed to compute p-values. Notably, U87MG-EGFRvIII-MTS1 cells are significantly more resistant to Iressa-mediated cell kill than U87MG-EGFRvIII cells. It is also noticeable that under the unstressed condition, U87MG-EGFRvIII-MTS1 cells had a reduced ability to form colonies compared to U87MG-EGFRvIII cells.