Epithin enhances transendothelial migration. (A) GFP-427 or GFP-427 Epi-KD cells were seeded on a confluent monolayer of MS1 cells. After incubation for 8 hours, the cells were fixed and stained for F-actin with rhodamine-conjugated phalloidin. Based on their morphology visualized by GFP and F-actin, round-shaped cells were counted as attached cells and spindle- or polygonal-shaped cells were counted as transmigrated cells. The representative images of attached (i-iii) and transmigrated (vii-ix,xiii-xv) cells in a sequence of the event are shown. Green represents the GFP signal (i,iv,vii,x,xiii,xvi); and red, the F-actin staining (ii,v,viii,xi,xiv,xvii) in the merged image (iii,vi,ix,xii,xv,xviii). Vertical images (iv-vi,x-xii,xvi-xviii) are also shown to visualize the height of the GFP-positive cells. (ix) *Actin stress fiber. (B) The mean values of the number of attached cells or transmigrated cells per field from 3 independent experiments are shown as bar graphs (left). The percentage of the number of transmigrated cells to the total number of transmigrated and attached cells is shown in each cell type (right). Error bars represent the SD. *P < .001.