Fig. 1. Effect of AZI on proliferation and anchorage-independent growth in AT2.1 prostate cancer cells. (A) 50 µg total cell lysate from AT2.1 cells stably transfected with pcDNA3 or pcDNA3-AZI constructs were analyzed for AZI and actin expression by western blotting. The stable AZI cell line used in these studies showed a fivefold increase in AZI expression relative to endogenous levels. (B) Proliferation of control () and AZI-overexpressing AT2.1 cells () in low serum (1% FBS). Cells were plated at a density of 1x10⁴ cells in 12-well plates and the total number of cells per well were counted. Error bars indicate standard error of the mean (s.e.m.). (C) Proliferation of control () and AZI-overexpressing AT2.1 cells () grown in high serum (10% FBS) was assessed as described above. (D) Phase-contrast micrographs of control and AZI-overexpressing AT2.1 cells comparing colony formation in 0.45% noble agar, as described in the Materials and Methods. Colonies were visualized after 2 weeks using 0.02% Giemsa stain. AZI-overexpressing cells formed more colonies with diameters greater than 0.5 mm. Similar results were seen with a second clone that expressed AZI at twice the wild-type level (data not shown).