Spy1 mediated colony formation is Cdk1 dependent. 293 cells transfected with the indicated constructs were (A) immunoprecipitated for HA tagged-Cdk1 and blotted for Myc and HA tag. n = 3 (B) imaged after 14 days in a soft agar assay. Transfections and protein levels were monitored by western blot analysis (inset). n = 3 (C) Quantification of colony number over three separate transfections. (D) Soft agar assays of cells transfected in triplicate with Myc-Spy1-TST in the presence or absence of Cdk dominant negative constructs (Cdk1-DN or Cdk2-DN). n = 3 Plates are imaged and quantified at day 14. Errors bars reflect SD between triplicate experiments. ANOVA with post hoc analysis using Tukey test was performed;* P ≤ 0.05, ** P ≤ 0.01.