S100A4 siRNA knock-down abolishes relaxin's effect on cell motility. Treatment of MDA-MB-231 cells with 200 nM S100A4 siRNA, but not with 200 nM non-silencing small interfering (si) RNA or Lipofectamine, resulted in down-regulation of S100A4 protein after 48 hours and 72 hours as shown here (a) after 48 hours. (b) When S100A4 siRNA treated MDA-MB-231 cells were subjected to 24 hours motility assays, relaxin was unable to increase cell motility. In contrast, cells transfected with non-silencing siRNA probe and cells treated with lipofectamine only were able to respond to 500 ng/ml rhRLN2 with an increase in motility similar to untreated cells. The results of three independent experiments are shown in the graph as mean ± SEM. One-way ANOVA analysis was performed. **p < 0.005 for an increase compared with equivalent control and #p < 0.005 for a decrease compared with the non-treated control (MDA).