Figure 5
Arf6 knockdown causes defect in directed cell migration of hLECs. (A) Tracking of cell migration during wound healing by time-lapse video microscopy. hLECs transfected with siRNAs for 48 hours were grown to the confluence, scratched, and stimulated with 200 ng/ml of VEGF-C. Images were obtained every 10 minutes for 20 hours after wounding. Each line represents the trajectory of an individual cell. Red and black lines indicate the euclidean distance over and less than 100 μm, respectively. Dotted lines indicate the boarder of the wound. Each experiment was performed four times, and at least 60 cells per experiment were analyzed. (B) Images shown in (A) were quantified for accumulated distance (left panel), euclidean distance (middle panel), and directionality (right panel), which was calculated by dividing euclidean distance by accumulated distance. (C) Representative phase contrast microscopic images of control and Arf6-knocked-down of hLECs (upper images) and immunostained images with anti-GM130 antibody (green), phalloidin (red), and DAPI (blue) (lower images) at the wounded edge (left panels). Arrowheads indicate elongated cells. Arrows indicate the migrating direction of the cell as decided by the location of Golgi. Images were quantified for cells with polarized Golgi (right panel). At least 200 cells per experiment were analyzed, and data were shown as means ± SEM from at least 3 independent experiments. Statistical significance was assessed using one-way ANOVA. NS, not significant, *P < 0.05, **P < 0.01. Scale bar, 25 μm.
