Actin arcs/DSF play a role in flattening U2OS cells. (A–F) Removing actin arcs/DSFs abolishes the lamella’s flatness. (A–D) Ventral (purple) and dorsal (blue-green) actin filament organization in a control U2OS cell (A), a cell treated with 20 µM blebbistatin for 2 h (B), a cell treated with myosin IIA–siRNA (C), and a cell treated with 10 µM Y-27632 for 2 h (D). Side views are x-z maximum projections from the yellow boxes. Arrows denote the transition between the flat portion of the cell (lamella) and the cell body. (E) Cell heights from five cells from the treatments in A–D. Arrows show the mean distance cell height rises above 2 µm (dotted lines). (F) Quantification of the distance from the leading edge at which cells become higher than 2 µm (n = 6 cells per condition from three separate experiments). Error bars show standard deviation. *, P < 0.001. Bars, 10 µm.