Fig. 2. Both cofilin and Arp2/3 are required for lamellipod extension. GFP-ßactin-expressing MTLn3 cells were either not injected (A-D), or microinjected with control nonimmune rabbit IgG (E-H), with anti-p34 function-blocking antibody (I-L), or with anti-cofilin function-blocking antibody (M-P, injected cell indicated with arrow). Cells were stimulated with EGF and time-lapsed to follow lamellipod extension and accumulation of F-actin at the leading edge. A, E, I and M show cells before the addition of EGF; B, F, J and N 90 seconds after EGF; and C, G, K and O 180 seconds after EGF. D, H, L and P show the difference in cell area before and after EGF (cell area before EGF: green; after EGF: red; overlap of areas: yellow). Some small protruding areas are indicated with arrowheads in K and O. Bar in A, 10 µm. (Q) Quantitation of cell area during lamellipod extension. Cell areas were measured every 10 seconds during time-lapse. Q shows the fold-increase in area after EGF stimulation of control nonimmune IgG-injected cells (n=15), anti-p34 (n=18) and anti-cofilin (n=17) antibody injected cells. (R) Dose-response for anti-p34 microinjected cells. Cells were co-injected with antibody and Texas Red-labeled dextran. Texas Red fluorescence was quantified as an indicator of relative amounts of antibody injected per cell, and measurements of cells were combined into four groups according to the fluorescent intensity of the cell (shown as normalized fluorescence intensity of Texas Red dextran, arbitrary units, a.u., below each column). The dose-response for each group is shown as average fold area increase 200 seconds after the addition of EGF, at which time cells have maximally extended. Measurements for uninjected cells are shown in the first column for comparison. (S) Dose-response for anti-cofilin injected cells. Error bars indicate ±s.e.m. In each case, both for R and S, there is no statistic difference between the last three columns, whereas the difference comparing the last three columns to the first two columns is statistically significant.