Figure 5

WT1 affects PCa cell migration. (A) A wound healing assay was performed after transfection with siWT1 RNA or RISC oligonucleotides or MOCK in PC3 cells. Migration potential was measured as described in methods. Monolayers were examined with an Olympus 1 × 70 microscope at 100X magnification and photographed at 0 and 16 hours after wounding. Representative images are shown and the bar represents 100 μM. (B) Six images per treatment were measured and analyzed by TScratch software (39) comparing open area at 16 hours to that at 0 hours. Values are presented as percent of open area of monolayers remaining at 16 hours compared to 0 hours and significance was determined by student t-test (**p ≤ 0.01,***p ≤ 0.001) in two independent experiments. (C) PC3 cells were transfected as described in (A) and placed in the upper chamber and allowed to migrate for 6 hours as described in methods. Representative images of hematoxylin stained membranes containing migratory cells are shown at 100x magnification. (D) Migratory cells were counted in six images per treatment and data are presented as average number of migratory cells per chamber. (E) LNCaP cells were transfected with pCMV4 or GFP/WT1 expression construct as described in Figure1. Transfected cells were placed in the upper chamber and allowed to migrate for 48 hours as described in methods. (F) Data are presented as per panel D and significance was determined by student t-test (**p ≤ 0.01,***p ≤ 0.001) comparing siWT1 RNA transfected relative to RISC transfected PC3 cells (D) and GFP/WT1 transfected relative to pCMV4 transfected LNCaP cells (F) in two independent experiments.