α-actinins are required for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation in spreading and polarized migrating cells. U2OS luc-shRNA (luc-sh) control cells (A) or U2OS α-actinin-shRNA (actinin-sh) cells (B) were plated onto fibronectin (Fn) coated glass coverslips and allowed adhesion for 15, 30, 120 min before fixation, immunofluorescence staining was performed for vinculin, actin stress fibers were stained with rhodomin–phallloidin, images were obtained by confocal fluorescence microscopy, scale bar represents 20 lm. Arrows in the images marked the dorsal stress fibers (DS), transverse arcs (TA), ventral stress fibers (VS), lamellipodium (LP in red), nascent adhesion (NA in green) and focal adhesion (FA).