Erg regulates the actin cytoskeleton and lamellipodia dynamics. (A) HUVECs were transfected with control-GB or Erg-specific GB for 40 hours. Cells were trypsinised and replated onto fibronectin-coated glass coverslips for 6 hours before being fixed, labeled and visualized for actin (green), Erg (red), and nuclear marker DAPI (blue). Scale bar, 20 μm. After Erg inhibition, actin stress fibers are redistributed into cortical rings around the cell periphery with loss of stress fibers across the cell body. In addition, Erg-deficient cells exhibit a pronounced angular shape. (B) HUVECs were transfected with actin-RFP and allowed to adhere to fibronectin-coated glass chamber slides for 24 hours before transfection with FITC-conjugated control or Erg-specific GB for an additional 24 hours. GeneBloc-transfected cells were identified by the presence of FITC fluorescence in the nucleus (not shown). In these cells, some actin stress fibers are present across the cell body, probably because of actin overexpression. Actin-RFP dynamics and lamellipodia formation were visualized using time-lapse confocal microscopy with images captured every 15 seconds over a period of 15 minutes. Representative images show dynamics of lamellipodia formation in control (top) and Erg cells (bottom). Kymographs show changes in actin-RFP fluorescence intensity at selected points across the cell membrane. In control cells, dynamic actin-rich lamellipodia and membrane protrusions are observed at multiple points around the cell membrane, while inhibition of Erg results in a dramatic reduction in the formation and collapse of actin-containing lamellipodia and the membrane appears static.