Overexpression of TRIP-Br2-HA confers anchorage-independent growth on soft agar and induces tumors in nude mice (nu/nu). (A) Anchorage-independent growth of NIH3T3vector-only, NIH3T3TRIP-Br1-HA and NIH3T3TRIP-Br2-HA was assessed by colony formation in soft agar. The error bars represent the standard deviations of three independent experiments performed in triplicates. PC3 cells were used as a positive control. V: Vector-only clones; R1: TRIP-Br1-HA-overexpressing clones; R2: TRIP-Br2-HA-overexpressing clones. (B) Upper panel: Results of a representative experiment in which NIH3T3TRIP-Br2-HA and NIH3T3vector-only fibroblasts were subcutaneously injected into the left and right flanks of nude mice, respectively. Lower panel: Average tumor ellipsoid volume over 25 days post-subcutaneous injection was calculated, and the animals were subsequently sacrificed. (C) Histological analyses of excised tumors indicated the presence of fibrosarcomas. (D) Western blot (Upper panel) and immunohistochemical analyses (Lower panel) of excised tumors showed expression of TRIP-Br2-HA. Immunopositive staining for TRIP-Br2-HA is represented by the brown color against the hematoxylin (blue) counterstain. Data was obtained from three independent experiments that were performed in triplicates.