Figure 3
STAT5b knockdown inhibits β1-integrin-mediated migration to fibronectin (FN). (a) The undersides of trans-well filters were coated with 10 μg/ml FN or vitronectin (VN) overnight at 4°C. BT-549 and MDA-MB-231 cells were placed in serum-free media in upper chambers, and 1% fetal bovine serum (FBS) (BT-549) or 10% FBS (MDA-MB-231) medium was placed in the lower chambers for FBS controls. Serum-free medium was placed in lower chambers for FN and VN conditions. Migration was allowed to proceed for 3 hours (BT-549) or 6 hours (MDA-MB-231) (n = 3). (b) BT-549 and MDA-MB-231 cells were pretreated for 1 hour at 37°C with 10 μg/ml β1-integrin-blocking antibody or DMSO control (con). Cells were plated in trans-well chambers in the presence of blocking antibody, and migration to 1% FBS (BT-549) or 10% FBS (MDA-MB-231) was measured. Student's t test was used to determine statistical significance (P < 0.05) between the following: BT-549: con and antibody (*) (n = 6); MDA-MB-231: con and antibody (*) (n = 4). (c) The undersides of trans-well filters were coated with 3 μg/ml FN overnight at 4°C, and migration assays were performed with siRNA-transfected cells as described in part a. One-way ANOVA with Tukey's post-test was used to determine statistical significance (P < 0.05) between the following: BT-549: siLuc and siSTAT5b FBS (*), siLuc and siSTAT5b FN (black circles); n = 4. MDA-MB-231: siLuc and siSTAT5b FBS (*), siLuc and siSTAT5b FN (black circles); n = 3.