Figure 3
EWS/WT1-KTS and EWS/WT1 + KTS increase anchorage independent growth and confer resistance to daunorubin induced apoptosis and cell cycle arrest following radiation. (A) 1x104 p53 wild-type, p53+/- or p53-/- MEFs expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS were plated in soft agar and the number of colonies greater than 2 mm counted after 14 days. Values are mean ± SEM of three independently generated and infected pools of MEFs for each genotype tested in three independent experiments. P values of less than 0.05 as determined by Student’s t-tests are shown. (B) Viability assay of cells treated with daunorubicin (0.5 μg/ml) for 24 hours. The data show the ratio of WST1 absorbance of treated cells to the same number of untreated cells. Values are mean ± SEM of three independent experiments. P values < 0.05 as determined by Student’s t-tests are shown. (C) Wild-type MEFS were irradiated with 10Gy gamma radiation and cell cycle analysis performed at the indicated time points by staining cells with hypotonic propidium iodide solution. Cell-cycle analysis was performed using FACS analysis and the Modfit software package. % of cells in G0/G1, S phase and G2 at baseline, 4, 12 and 24 hours post radiation are shown. Values represent mean of three independent experiments. ** represent p values <0.05 in the comparison of % of cells in S phase and G1 compared to eGFP controls, as determined by Student’s t-tests.