Rac3 regulates integrin signaling at invadopodia through CIB1. (A, images) Images of control (top) or Rac3 siRNA (bottom)–treated MDA–MB-231 cells plated on gelatin, starved, and stimulated with EGF for 0 min (left) or 5 min (right). Cells were stained for active or total integrin as well as cortactin and Tks5 to denote invadopodia. Yellow arrows indicate invadopodia. (A, graph) The ratio of active/total integrin at invadopodia in control or Rac3 siRNA–treated cells. n ≥ 25 invadopodia from ≥5 cells for each condition; three independent experiments. (B) Exogenous Mn²⁺ treatment rescues invadopodia lifetimes in Rac3-depleted cells. n ≥ 98 invadopodia from ≥3 cells for each condition; three independent experiments. (C) Images of endogenous CIB1 at invadopodia (cortactin/matrix) in MTLn3 cells. (D) Endogenous CIB1 accumulation at invadopodia in MTLn3 cells stimulated with 5 nM EGF for the indicated times or plated overnight on gelatin (steady state). n ≥ 50 invadopodia from ≥25 cells for each condition; three independent experiments. (E) MTLn3 cells transfected with control (top) or CIB1 siRNA (bottom) and plated on 405 nm fluorescent gelatin overnight. (E, top) Normalized, mean degradation area/field. n ≥ 10 fields for each condition; three independent experiments. (E, bottom) Mean number of invadopodia/cell. n ≥ 50 invadopodia from ≥25 cells for each condition; three independent experiments. Western blot of cell lysates of control or CIB1 siRNA–treated MTLn3 cells blotted for CIB1 or β-actin with quantification. (F) Invadopodia lifetime when CIB1 is depleted. n ≥ 30 invadopodia from ≥10 cells for each condition; three independent experiments. (G) Images of endogenous Rac3 localization at invadopodia in control or CIB1-depleted cells. Quantification of number of invadopodia/cell positive for endogenous Rac3 in MTLn3 cells treated with control or CIB1 siRNA. n ≥ 75 invadopodia from ≥27 cells for each condition; three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.01. All error bars are SEM.