PMC

STAT5b knockdown negatively affects directionality during wound closure. Wounds were made in confluent monolayers of siRNA-transfected BT-549 cells, and time-lapse microscopy was used to track cell movement into the wound over a 6-hour period. (a) Progressive line images tracking single cell nuclei of cells along the wound edge. Two independent experiments are shown. (b) Migration rate (μm/h) was calculated by dividing the length of the migration path by the total movie time. (c) Directional persistence (D/T) was determined as the net displacement (D) divided by the total length of the migration path (T). (b, c) Values from at least four independent experiments were used for calculations (siLuc, n = 24; siSTAT5b, n = 32). Student's t test was used to determine statistical significance (p < 0.05) between siLuc and siSTAT5b (*). Comparisons of migration rate were not statistically significant.

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.

Expression of wild-type, Y699F-, or dominant-negative STAT5b rescues migration, but expression of R618K-STAT5b does not. (a) Domain structure of the STAT5b protein depicting the amino terminus (N-term), DNA-binding domain (DBD), Src homology 2 domain (SH2), transactivation domain (TAD), and the location of the conserved tyrosine residue, Y699, and arginine residue, R618. (b) MDA-MB-231 breast cancer cells were transfected with control siRNA to luciferase (siLuc) or siRNA specific to STAT5b (siSTAT5b siGENOME SMARTpool oligonucleotide #3) alone or in the presence of HA-tagged wild-type (wt-STAT5b), Y699F (YF-STAT5b), dominant-negative (dn-STAT5b), or R618K (RK-STAT5b) STAT5b constructs engineered to be immune to siRNA knockdown. Seventy-two hours after transfection, trans-well assays were performed for 6 hours, as described in Figure 1. Results are graphed as relative migration, compared with siLuc control. One-way ANOVA with Tukey's post-test was used to determine statistical significance (p < 0.05) between the following: siLuc and siSTAT5b (*), siSTAT5b and siSTAT5b + wt-STAT5b (black circles), siSTAT5b and siSTAT5b + YF-STAT5b (black circles), siSTAT5b and siSTAT5b + dn-STAT5b (black circles); n ≥ 4. Comparison of siSTAT5b and siSTAT5b + RK-STAT5b was not statistically significant. Whole-cell lysates from transfected cells were immunoblotted with antibodies specific for STAT5b, HA, or β-actin as a loading control.

STAT5b-knockdown cells exhibit multiple protrusions with increased retraction. BT-549 cells were transfected with siRNA (siLuc or siSTAT5b), GFP-speckle-actin, and mKO-paxillin. Seventy-two hours after transfection, cells were plated on 3 μg/ml fibronection (FN) for 20 to 30 minutes, and TIRF time-lapse microscopy was performed. Images were taken every 3 seconds for 5 minutes at 60× magnification. (Line indicates 10 μm). (a) Still images representing actin and paxillin staining in control (siLuc) and STAT5b-knockdown (siSTAT5b) cells at start of filming (0 min) and end of filming (5 min). (b) Enlarged images depicting boxed areas from part a. Arrows identify single focal adhesions at each time point. (c) Protrusion rate (μm/min) was calculated as the length of the protrusion divided by the total time of the movie for at least three independent experiments (siLuc, n = 16; siSTAT5b, n = 13). Student's t test was used to determine statistical significance (P < 0.05) between siLuc and siSTAT5b (*).

STAT5b knockdown inhibits breast cancer cell migration. (a) BT-549 or MDA-MB-231 breast cancer cells were transfected with no siRNA (con), control siRNA for luciferase (siLuc), siSTAT5b SMARTpool (BT-549 cells), or siSTAT5b SMARTpool duplex #3 (MDA-MB-231 cells). Seventy-two hours after transfection, cells were plated in serum-free media in trans-well chambers. Media containing 1% fetal bovine serum (FBS; BT-549) or 10% FBS (MDA-MB-231) were placed in the lower chambers. After 3 hours (BT-549) or 6 hours (MDA-MB-231), cells were fixed, stained with crystal violet, and the number of migratory cells was counted. Results are graphed as the average number of migratory cells per field ± SEM. One-way ANOVA with Tukey's post-test was used to determine statistical significance (P < 0.05) between the following: BT-549: con and siSTAT5b (*), siLuc and siSTAT5b (black circles); n = 5. MDA-MB-231: con and siSTAT5b (*), siLuc and siSTAT5b (black circles); n = 4. (b) BT-549 breast cancer cells were transfected and plated in serum-free media in trans-well chambers for 3 hours. Media containing varying concentrations of FBS (0.1% to 10%) were placed in the lower chambers. One-way ANOVA with Tukey's post-test was used to determine statistical significance (P < 0.05) between the following: con and siSTAT5b (*), siLuc and siSTAT5b (black circles); n = 3. (c) MDA-MB-231 cells were transfected with no siRNA (con), control siRNA for luciferase (siLuc), or siRNA specific to STAT5a/b (siSTAT5b siGENOME SMARTpool), STAT5a (siSTAT5a custom oligonucleotide), or STAT5b (siSTAT5b siGENOME SMARTpool oligonucleotide 3), and trans-well migration assays were performed as described. One-way ANOVA with Tukey's post-test was used to determine statistical significance (P < 0.05) between the following: con and siSTAT5a/b (*), con and siSTAT5b (*), siLuc and siSTAT5a/b (black circles), siLuc and siSTAT5b (black circles); n ≥ 5. (a and c) Whole-cell lysates from siRNA-transfected cells were immunoblotted with antibodies specific for STAT5a, STAT5b, and β-actin as a loading control.