FIGURE 2.

Silencing Ets2 induces telomerase inhibition-dependent breast cancer cell death. A, effects of silencing Ets1 and Ets2 on breast cancer cell proliferation. MCF-7 cells at 1 × 10⁵ cells per well in 24-well plates were transfected with four different shRNA expression plasmids individually as indicated and cell numbers were monitored on each day of the transfection for 3 days. The results were mean ± S.D. of three determinations of cell numbers from three experiments. B, staining of apoptotic cells in MCF-7 cell cultures. Cells were transfected for 48 h with different shRNA expression plasmids as indicated and labeled with ApopTaq for DNA breaks. Nuclear total DNA was stained with Hoechst. Micrographs were from fluorescence microscopy at ×10 magnification. C, effect of silencing Ets1 or Ets2 by specific siRNAs on apoptosis. Cells were stained with Annexin V and propidium iodide, and analyzed by FACS. Annexin V and propidium iodide double positive cells in the upper right panel of B were expressed as a percentage of total cells. D, re-establishment of hTERT gene expression in Ets2-silenced cells. Cells with Ets2 gene silencing constructs were transfected with HA-hTERT for 48 h. Cell lysates were probed with specific antibodies for Ets2 and hTERT with β-tubulin as control. E, effects of hTERT overexpression and Ets2 transduction on apoptosis induced by silencing Ets2. MCF-7 cells stably transfected with the different shRNA expression plasmids as indicated were transduced with hTERT gene expression plasmids, Ets2 fusion protein, or GST control for 24 h. Cells were stained with propidium iodide followed by FACS. The percentages of cells with a hypodiploid (sub-2N) DNA content indicative of apoptosis are shown. F, effects of Ets2 siRNA on apoptosis in cultured cells expressing hTERT. Cells with or without recombinant GFP-hTERT or GFP only were transfected with Ets2 siRNA for 48 h followed by FACS. Data are representatives of three experiments.