Fig. 3. Activation of BRCA2 gene silencer in MDA-MB-468 cells by the overexpression of the SLUG protein.

A, a Northern autoradiogram showing overexpression of SLUG in the doxycycline-induced (Dox +) cells. An ~0.8 kb DNA containing full-length human SLUG coding sequence was used as probe. The size of SLUG mRNA is 2.2 kilo nucleotide. 28 S rRNA is shown as a loading control. B, a Western blot showing induced expression of SLUG protein in the recombinant MDA-MB-468 cells. C, real-time RT-PCR data showing an increase in the SLUG mRNA level and a decrease in the BRCA2 mRNA level in the presence of doxycycline. D, an electrophoretic mobility shift assay (EMSA) showing the doxycycline-inducible binding of protein in recombinant MDA-MB-468 cells. Nuclear proteins (NE) were isolated from wild-type (*) or recombinant (Dox − or Dox +) MDA-MB-468 cells. The probe was ³²P-labeled 221-bp wild-type silencer DNA. E, the functional activity of the silencer in the uninduced (No Dox) or doxycycline-induced (With Dox) recombinant MDA-MB-468 cells. Cells were transfected with pGL3-P, pGL3-PS, or pGL3-PS* (which has the E2-box-mutated silencer sequence instead of the wild-type silencer) plasmid DNA. Data are presented as mean (n = 12) ± S.E. The differences between the luciferase activities in the cells (transfected with pGL3-PS construct) treated with and without doxycycline were statistically significant (p < 0.0001). RLU, relative light units.