Figure 8.
E6AP Promotes Metastatic Phenotype via Suppression of NDRG1 that Can Be Restored with Thiosemicarbazones
(A) DU145 stably transduced with shNDRG1 (+) or a shRNA control (−) were transiently transfected with a siRNA against E6AP (siE6AP +) or a siRNA control (−). At 72 h post-transfection cells were collected for immunoblot.
(B) Cells were seeded in transwells and allowed to migrate for 24 h as described in (A). Invaded cells were stained with crystal violet (left) and quantified at A570 after extraction of the dye (right). Each experiment was repeated three independent times. *p < 0.05, ***p < 0.001, unpaired t test.
(C) DU145 cells were treated with DFO (positive control; 250μm), Bp2mT (negative control analogue; 5μm), Dp44mT, DpC, or Bp2mT (5μm each) for 24 h, and the levels of E6AP and NDRG1 were determined by Western blotting. β-actin was used as a loading control; densitometry quantification of the expression derived from triplicates of one of three independent experiments is shown. **p < 0.01, unpaired t test.
(D) The effect of the compounds on cell migration was determined by transwell assays. Invaded cells were stained with crystal violet (left) and quantified at A570 after extraction of the dye (right). Each experiment was performed in duplicates and repeated three independent times. Quantification of a representative experiment is shown as mean ± SD. Scale bar represents 100 μm.