Vav2 and βPIX GEFs work in concert to regulate Rac3 at invadopodia. (A) Image of EGFP-Vav2 localized to invadopodia core in MDA–MB-231 cells plated on gelatin for 6 h or MTLn3 cells plated on gelatin for 16 h. The bottom graph shows quantification of the percentage of Vav2-positive invadopodia in the indicated cell line. (B) Image of EGFP-βPIX localized to the ring (top) or core (bottom) of invadopodia in MDA–MB-231 cells plated on gelatin for 6 h or MTLn3 cells plated on gelatin for 16 h. White arrows indicate invadopodia. Quantification of the percentage of βPIX-positive invadopodia in the indicated cell line. (C, left) Normalized mean degradation area per field in MDA–MB-231 cells with control, Vav2, or βPIX siRNA and plated on 405 nm fluorescent gelatin for 6 h. n ≥ 10 fields for each condition; three independent experiments. (C, right) The mean number of invadopodia per cell for the same conditions. n ≥ 50 invadopodia from ≥25 cells for each condition; three independent experiments. Western blot of cell lysates of control, Vav2 (top), or βPIX (bottom) siRNA–treated MDA–MB-231 cells blotted for Vav2, βPIX, or β-actin with quantification. (D, right) The percentage of invadopodia with high core (black bars) or high ring (gray) Rac3 activity in MDA–MB-231 cells with control, Vav2, or βPIX siRNA. n ≥ 18 invadopodia in ≥5 cells; three independent experiments. Data were analyzed using a one-tailed paired Student’s t test. (D, left) Whole-cell intensity quantification of Rac3 activity in control, Vav2, or βPIX-depleted cells. Representative images of Rac3 activity in control (top), Vav2 siRNA (middle), or βPIX siRNA (bottom)–treated cells. Dashed lines indicate the invadopodium core and ring. All Rac3 activity images are scaled identically. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. All error bars are SEM.