Knockdown of epithin reduces metastatic ability of 4T1 cells. (A) The levels of epithin in 4T1 cell line (4T1) and its derivatives (A7, D5, B3, C5, and G3) were measured by Western blot with antiepithin mAb5 antibody. An equal amount of protein loading was confirmed by antitubulin blot (not shown). (B-C) The number of transmigrated 4T1 cells through MS1-confluent Boyden chamber was counted and plotted. Each dot in the graph represents the number of transmigrated cells in a microscopic field. The add-back clone AB-B3 was generated by stably transfecting pcDNA3.1/Matriptase (human ortholog of mouse epithin) into B3. (D) Each of 4T1 clones was injected into tail veins of 6 Balb/c mice. After 2 weeks, mice were killed and their lungs were stained with Indian ink. Representative lung images of each group are shown. (E) The numbers of tumor nodules on the lungs were counted and plotted. (B-E) Data are mean plus or minus SE. *P < .001. **P < .01.