Spheres generated from 293T cells are resistant to therapy. A) Clonogenic/mammosphere formation assay of 293T cells cultured under 2D or 3D promoting culture conditions. Cells in 3D are more resistant to radiation than cells grown in 2D. Cells were grown in 2D or 3D culture conditions and irradiated with single, increasing doses (0, 2, 4, 6 Gy) of radiation. B) Upregulation of genes implicated in radioresistance including β-catenin, Notch and Survivin in 3D cells 48 hours after 4 Gy of irradiation. C) 3D cultured cells are also more resistant to chemotherapeutic drugs like retinoic acid compared to 2D. Similar results were obtained with Taxol (data not shown). Indicated concentrations of drugs were added to the media until the number of colonies or spheres were counted.