Figure 4.

AR is required for androgen-induced motility and invasiveness of MDA-MB453 cells. MDA-MB453 cells were transfected with AR siRNA or non-targeting siRNA, using Lipofectamine^TM 2000. siRNA Alexa Fluor 488 was included to identify transfected cells. After transfection, the cells were made quiescent for 24 h and then used for migration (a) or invasion (b) assays. In (a), migration assay was done in Boyden’s chambers pre-coated with collagen. In (b), invasion assay was done in Boyden’s chambers pre-coated with growth factor-reduced and phenol red-free Matrigel. In both assays, the indicated compounds (R1881 at 10 nM, bicalutamide at 1μM, S1 peptide at 10 nM and cytosine arabinoside at 50 μM) were added to the upper and the lower chambers. In (a), transfected cells were allowed to migrate for 24 h and then fixed in 4% paraformaldehyde for 20 min. In (b), transfected cells were allowed to invade for 36 h. Alexa Fluor 488-migrating (a) or invading (b) cells were finally scored and counted using a DMBL (Leica) fluorescent microscope equipped with HCPL Fluotar 20x objective in 30 random microscopic fields. In (a,b), results were collected and expressed as fold increase. Means and SEMs are shown. n represents the number of experiments. *p < 0,05 for the indicated experimental points versus the corresponding untreated control. In (c), lysates from MDA-MB453 cells transfected with AR siRNA or non-targeting siRNA were prepared. Proteins were separated by SDS-PAGE and then analyzed by Western blot, using the antibodies against the indicated proteins.