Rac3 is enriched at invadopodia and required for matrix degradation. (A) Endogenous Rac3 accumulated at invadopodia in MTLn3 or MDA–MB-231 cells plated on a 405-nm fluorescent gelatin matrix. Invadopodia are denoted by cortactin colocalization with spots of matrix degradation. (B) Quantification of endogenous Rac3 accumulation at invadopodia in MTLn3 cells stimulated with 5 nM EGF for the indicated times. n ≥ 30 invadopodia from ≥20 cells for each condition; three independent experiments. (C, images) MTLn3 cells transfected with control (top) or Rac3 siRNA (bottom) and plated on 405 nm fluorescent gelatin overnight. (C, bottom left) Normalized, mean degradation area/field. n ≥ 10 fields for each condition; three independent experiments. (C, bottom right) Mean number of invadopodia/cell. n ≥ 50 invadopodia from ≥25 cells for each condition; three independent experiments. Western blot of cell lysates of control and Rac3 siRNA–treated MTLn3 cells blotted for Rac3 and β-actin with quantification. (D) Rescue of siRNA Rac3 or Rac1 depletion by expression of WT or DN Rac3 or Rac1, respectively. (D, top) Degradation area/cell. n ≥ 20 cells for each condition; three independent experiments. (D, bottom) Mean number of invadopodia/cell. n ≥ 25 invadopodia from ≥20 cells for each condition; three independent experiments. (D, right) Western blot of cell lysates of control and Rac1 siRNA–treated MTLn3 cells blotted for Rac1 and β-actin, with quantification below. (E, left) Mean invadopodia lifetime. n ≥ 30 invadopodia from ≥10 cells for each condition; three independent experiments. (E, right) Same data shown as histogram with bins corresponding with invadopodia lifetimes in 20-min intervals. (F) Fold change in lifetime of invadopodia containing mCherry-Rac3 in MTLn3 cells. n = 29 invadopodia from ≥10 cells for each condition; three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01. All error bars are SEM.