Figure 2. Induced expression of hOsa2 results in impaired cell growth and accumulation of cells in the G₁ phase.

(A) Nuclear extracts were prepared from 16eC3 cells uninduced (lanes 1, 4 and 7) or induced to express FLAG–hOsa2 (lanes 2, 5 and 8: 0.01 ng/ml Dox; lanes 3, 6 and 9: 0 ng/ml Dox). Shown is an immunoblot probed sequentially with antibodies against FLAG, BRG1 and Sp1. The molecular mass in kDa is indicated on the left-hand side. (B) 16eC3 cells were plated at 5×10⁵ cells per plate in 10 ng/ml Dox and induced the next day (day 0, Dox 0, 0 ng/ml) or left uninduced (day 0, Dox 10, 10 ng/ml). The total cell number was determined each day for a period of 7 days. Cells induced to overexpress hOsa2 showed a substantial and increased reduction in cell number compared with uninduced cells. The cell number did not change in the control cells that were treated similarly with Dox (results not shown). Cells that were induced on day 0, but were refed in a medium containing 30 ng/ml Dox on day 2 (Dox 0–30) to shut down hOsa2 expression, showed a partial recovery in cell number. Similar results were obtained with an independent cell line 16eC8. (C) Uninduced and induced 16eC3 cells were subjected to DNA staining and flow cytometry. Upon removal of Dox, 16eC3 cells and 16eC8 cells (results not shown) accumulated in the G₀/G₁ phase of the cell cycle. The control cells showed no change in cell cycle distribution in the same experiment (results not shown).