FIGURE 8.
Knockdown of endogenous Ang2 inhibits the Akt-Bcl-2 signaling and decreases cell survival, migration, and inhibits cell apoptosis of MDA-MB-468 and SK-BR-3 cells in vitro. A, IB analyses: knockdown of endogenous Ang2 by Ang2.siRNAs (A) or a control (C) abrogated Ang2-indueced p-AktSer473 and protein expression of Bcl-2 in MDA-MB-468 and SK-BR-3 cells. Total proteins of Akt and β-actin were used as loading controls. Cell survival (B), cell migration (C), and apoptosis (D) in the cells culture under serum-starvation. Ang2#1 cells were separately transfected siRNA pools for control (C) or Ang2 (A). 48-h later, the cells were incubated with serum-free/phenol red-free medium (serum-starvation) for 4 days. The numbers of survival cells were analyzed by trypan blue dye exclusion, counted and normalized to the cell number of each group seeded on day 0 (B). C, transfected MDA-MB-468 or SK-BR-3 cells were seeded into upper wells of in a Boyden chambers with 1 × 105 cell per well. Various cells were allowed to migrate through the 12 μm pore size membranes precoated with gelatin for 12 h at 37 °C. After the membrane was fixed and stained, non-migrating cells were removed. The number of migrating cells was quantified under a microscope as we previously described (10). Impact on cell migration was examined. D, cell apoptosis was analyzed using a cell death detection ELISA kit. Data of B–D were statistically analyzed using t test. *, p < 0.001 and error bars: mean ± S.D. The results of A–D are representative from three independent experiments with similar results.