FIG. 6.

Freud-1/Aki1 gene silencing induces apoptosis and enhances the sensitivity to antitumor drugs. (A) 293T cells were transfected with nonsilencing control siRNA, Freud-1/Aki1-1 siRNA (circles), or Freud-1/Aki1-2 siRNA (triangles). After transfection for 48, 72, or 96 h, the MTT assay was performed, as described in Materials and Methods. Error bars represent standard deviations of triplicate experiments. (B) 293T cells were transfected with nonsilencing control siRNA (open columns) or Freud-1/Aki1-1 siRNA (solid columns). After transfection for 48, 72, or 96 h, the number of apoptotic sub-G₁ fractions was determined by flow cytometric analyses, as described in Materials and Methods. (C) 293T cells were transfected with nonsilencing control siRNA (open columns) or Freud-1/Aki1-1 siRNA (solid columns). After transfection for 72 h, cells were treated with DMSO, 10 μM Taxol, 10 μg/ml VP-16, or 10 μg/ml CPT. After incubation for an additional 24 h, the MTT assay was performed, as described in Materials and Methods. Error bars represent standard deviations of triplicate experiments. (D) 293T cells were transfected with nonsilencing control siRNA (−) or Freud-1/Aki1-1 siRNA (+). After transfection for 72 h, cells were treated with DMSO (lanes 1 and 2), 2.5 μM Taxol (lanes 3 and 4), 10 μg/ml VP-16 (lanes 5 and 6), or 10 μg/ml CPT (lanes 7 and 8). After incubation for an additional 24 h, cell lysates were electrophoresed and immunoblotted with the indicated antibodies.