FIGURE 1.

Mutation of Lys-99 abrogates the EN ability to transform NIH3T3 cells. A, shown is a soft agar colony formation assay. NIH3T3 cells expressing the indicated constructs were seeded in soft agar medium in triplicate. After 2 weeks of incubation, the single cells and colonies were counted. Results with S.E. are given as percentage of colonies formed per total number of events counted per sample. B, shown is immunoblot analysis of cleaved PARP to probe detachment-induced cell death. Equal numbers of cells were seeded in poly(2-hydroxy-methacrylate)-coated plates and grown as suspension cultures for 48 h. Spheroids were collected and analyzed by immunoblots. Total PARP antibody was used to detect cleaved PARP levels (lower band as indicated). NTRK3 antibody was used to detect EN expression and total Akt was used as loading control. Cleaved-PARP levels were quantified by ImageJ software and normalized to total Akt (loading control). C, pictures depict the spheroid sizes formed by the NIH3T3 cells expressing the indicated constructs in suspension cultures for 48 h. Spheroid sizes were measured using ImageJ software. A two-tailed Student's t test was used for statistical analysis. **, p < 0.005.