Spy1 stable protein induces anchorage independent growth. (A) 293 cells were transfected with different concentrations of Myc-Spy1-WT, Myc-Spy1-TST or 30 μg of empty PCS3 vector control. Soft agar assay was carried out and plates photographed and quantified on day 14. Foci were averaged over 3 separate transfections for each experiment. n = 3 (B) Western blot analysis of one representative experiment; densitometry in lower panel. n = 3 (C) Representative foci after 14 day soft agar assay visualized using light microscopy. Ras-V12 is transfected as a positive control. n = 3. (D) Total numbers of foci were counted over 3 separate plates using separate transfections for each experiment. n = 3. Error bars reflect SE between triplicate experiments. t test was performed;** P ≤ 0.01. (E) Western blot analysis of experiments in Figure. 1C & D. Quantified using densitometry followed by normalization for Actin levels. (A-E) Error bars reflect SE between triplicate experiments. t test was performed;* P≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.