Effects of RNAi knockdown of <i>Zip4</i> and/or forced expression of <i>Zip4</i> on the migration of Hepa and MCF-7 cells.

<p>Hepa cells expressing <i>Zip4</i> RNAi (<b>Z4 RNAi</b>) or control RNAi (<b>Con RNAi</b>) from lentivirus vectors were purified for GFP expression by FACS. Cells were seeded onto fibronectin-coated Transwell inserts and allowed to migrate for a period of 16 h. Non-migratory cells were removed and cells that had migrated through the membrane were fixed and stained with crystal violet. Cell migration was visualized by microscopy (<b>Part A</b>) and quantified by measuring the absorbance of cell lysates at 595 nm (<b>Part B</b>). In parallel, Hepa cells expressing <i>Zip4</i> RNAi or control RNAi were transfected with a human <i>Zip4</i>-HA expression vector (<b>Z4HA</b>), serum starved overnight and then seeded onto Transwell inserts and allowed to migrate for 16 h. Migratory cells were visualized and quantified as described above. ** Indicates a significant difference from control RNAi and/or <i>Z4</i> RNAi, left two bars (P<0.001) and # indicates a significant difference from control RNAi and/or <i>Z4</i> RNAi/<i>Z4</i>HA (P<0.001). <b>Part C:</b> MCF-7 cells were transfected with an expression vector for mouse <i>Zip4</i>-HA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013158#pone.0013158-Wang1" target="_blank">[33]</a> (<b>Z4HA</b>), or exposed to transfection reagent without vector (<b>control</b>), and then seeded onto fibronectin-coated Transwell inserts. Cells were allowed to migrate for a period of 16 h, non-migratory cells were removed and cells that had migrated through the membrane were fixed and stained. Cell migration was quantified by counting the numbers of cells in 5 randomly-chosen fields of view per membrane (n≥3 for each treatment) and results are plotted as the mean cell migration (percent of control ± S.D.). ** Indicates a significant difference between control and <i>Z4</i>HA transfected MCF-7 cells (P<0.001).