Arf6 controls the Cdc42-mediated polarity signaling pathway and cell orientation during migration. Astrocytes were nucleofected with the indicated siRNA or microinjected with the indicated constructs. (A) Immunostaining of Par6 and aPKC in migrating astrocytes 4 h after wounding. (right) Higher magnification views of the boxed regions are shown. Graphs plotting the fluorescence intensity of Par6 and aPKC measured along lines drawn perpendicularly to the leading edge of three different siCTL (blue)- and siArf6-2 (red)–transfected cells are shown below the corresponding images. Histogram showing the percentage of wound-edge cells with a leading edge accumulation of Par6 (black bars) and aPKC (gray bars). (B) Immunostaining of Thr⁴¹⁰-phosphorylated aPKC in control or Arf6-depleted astrocytes 4 h after wounding. (right) Higher magnification views of the boxed areas are shown. The histogram shows the percentage of cells with an accumulation of phospho-aPKC at the leading edge. (C) Immunostaining of APC (red) and tubulin (green) in migrating astrocytes 8 h after wounding. (right) Histogram shows the percentage of wound-edge cells with APC clusters at microtubule plus ends. (D) Fluorescence images of the microtubules (green), centrosome (red), and nucleus (blue) in nucleofected migrating astrocytes 8 h after wounding. (E) Centrosome and Golgi reorientation 8 h after wounding. (F) Immunostaining of tubulin in migrating astrocytes 16 h after wounding. Data are shown as mean ± SEM of three independent experiments totalizing >300 cells. The dotted lines indicate the direction of the wound. Bars, 10 µm.