Figure 6.

Fn14 depletion in MCF7/HER2-18 cells reduces HRG1-β1-stimulated cell migration and invasion. (A) The MCF7/HER2-18 control shRNA-1A and Fn14 shRNA-448 cell lines were serum-starved overnight (0.5% FBS) and then either left untreated or treated with HRG1-β1 (50 ng/ml) for the indicated time periods. Cells were harvested and Fn14 and tubulin expression analyzed by Western blotting. (B) MCF7/HER2-18 control and Fn14 shRNA cell lines were treated with HRG1-β1 (200 ng/ml) and migration capacity was compared using the scratch wound assay. Wound width was calculated at 0 and 24 h and the difference plotted as percent wound closure. The values shown are mean +/− SEM of three experiments. **P< 0.01 compared to control shRNA-1A cells by Students t test. (C) MCF7/HER2-18 control and Fn14 shRNA cell lines were treated with HRG1-β1 (200 ng/ml) and invasive capacity was compared using the Matrigel invasion assay. HRG1-β1-stimulated cell invasion was quantitated as described in Materials and Methods. The values shown are mean +/− SEM of three experiments. **P< 0.01 compared to control shRNA-1B cells by Students t test. (D) Fn14 shRNA-448 cells, stably transfected with vector or Fn14-myc plasmid, were analyzed for Fn14-myc and tubulin expression by Western blotting. (E) Vector or Fn14-transfected Fn14 shRNA-448 cells were treated with HRG1-β1 (200 ng/ml) and invasive capacity was quantitated as described in Materials and Methods. The values shown are mean +/− SEM of three experiments. **P< 0.01 by Students t test.