Figure 5. DNmDia reduced directional migration.

A) Subconfluent cultures of 10T1/2 cells were transfected with mCherry-EV (Control) or mCherry-DNmDia. Live-cell imaging was used to monitor random (unstimulated) migration of 8 cells from each group for 8h. Individual cell migration paths from a relative starting point (0,0) are shown and net distance traveled was quantified using ImageJ. B) Confluent cultures of 10T1/2 cells expressing either mCherry-EV (Control) or mCherry-DNmDia were subjected to scratch wound with a P1000 pipette tip and then placed on an inverted microscope equipped with a heated, humidified, and O₂/CO₂ perfused stage. Pictures taken every 5 minutes for 24 hr were assembled into movies using Quicktime. Nine cells from each group, starting at the front of the scratch wound, were tracked. The averages of their displacement towards the wound was averaged at every time point and plotted for 24 hr. Nine cells near the wound edge from each group were tracked for 24 hr. Graphs are oriented such that migration toward the scratch wound is indicated by a positive value on the x-axis. C) Average displacement of cells from “B” were plotted over time +/− SEM. Repeated measurement one-way ANOVA analysis followed by Bonferroni’s post-hoc test for individual significance demonstrated that DNmDia expression significantly inhibited migration toward the scratch wound (p <0.05). D) 10T1/2 cells expressing either mCherry or mCherry-DNmDia were subjected to scratch wound for 8 hrs, fixed, and lamellipodia orientation toward or away from the wound was scored in at least 100 cells from three separate experiments. E) 10T1/2 cells expressing either mCherry or mCherry-DNmDia were subjected to scratch wound for 8 hrs, fixed, and probed for α-Tubulin and stained with DAPI to mark MTOC orientation. At least 100 cells were counted in three separate experiments. F) Primary rat aortic SMCs expressing either mCherry or mCherry-DNmDia were subjected to scratch wound for 8 hrs, fixed, and immunostained for Golgi marker (GM)-130 and DAPI to mark orientation. At least 100 cells from three separate experiment were counted. *p <0.05.