Figure 7

miR-221/222 and -503 overexpression increases, and TIMP2 knockdown decreases, cell motility. (A,B) Representative images of migrated (top panels) and invaded (bottom panels) cells for MCF-7 vector (A) and p95HER2 (B) cells. 48 h after induction and transfection with negative control miR (neg. miR), miR-221, miR-222, or miR-503, cells were resuspended in media containing 1% FBS and seeded for migration (non-coated) and invasion (coated) chambers placed in media containing 10% FBS for 24 h. Migrated/invaded cells were counted (20–80 images pr. membrane). Scale bar: 20 µM. (C,D) Quantification of migrated MCF-7 vector (C) and p95HER2-expressing cells (D). (E,F) Quantification of invaded MCF-7 vector- (E) and p95HER2 expressing cells (F). Data in (C,F) is shown as mean values relative to neg. miR +/− S.E.M., n = 3 for each cell line and condition. *Significantly different from neg. miR, p < 0.05, One-way ANOVA followed by Dunnet’s post-test. (G,H) Relative protein level of TIMP2 in vector (G) and p95HER2 (H) after knockdown of TIMP2. 48 h after induction and transfection with mock or TIMP2 siRNA, cells were lysed and subjected to Western blotting. Representative blots are shown, with β-actin as loading control. Graphs are mean values with S.E.M. error bars, n = 2–3 per condition. M: mock siRNA, T: TIMP2 siRNA. (I–L) Representative images of migrated (top panels) and invaded (bottom panels) cells for vector (I) and p95HER2 (L). 48 h after induction and transfection with mock or TIMP2 siRNA, cells were resuspended and seeded for migration/invasion as described in A + B. Scale bar: 20 µM. (J,K) Quantification of migrated MCF-7 vector (J) and p95HER2-expressing cells (K). (N,M) Quantification of invaded MCF-7 vector- (N) and p95HER2-expressing cells (M). Data in J-K and N-M is shown as mean values relative to mock +/− SEM, n = 3 (vector), 4 (p95HER2). **Significantly different from mock, p < 0.01, Student’s t-test.