Kindlin2 function in invadopodia formation is regulated by phosphorylation. (A) Effect of expressing dominant-negative phosphomutants of kindlin2 on invadopodia formation in MDA-MB-231 cells invading HDFC in the presence of serum. Means ± SEM of 100 cells/condition with 3–4 independent repeats. (B) Quantification of the effects of kindlin2 knockdown with single duplex siRNA and rescue with siRNA-resistant wild-type kindlin2 (siR-wt) or its mutants S159/181/666A (3A) and S159A on invadopodia formation in MDA-MB-231 cells invading HDFC. Values are means ± SEM of ∼100 cells/condition with three repeats. (C) Effect of expression of kindlin2 phosphomimetic mutants on invadopodia formation in carcinoma cells on globular collagen with no serum. Means ± SEM of 100 cells/condition with 3–4 repeats. (D) Effect of kindlin2-GFP dominant-negative mutants on invadopodia formation in MDA-MB-231 cells invading the HDFC matrix. Cells were cultured on HDFC for 3 h, fixed, and immunolabeled for actin and cortactin to identify invadopodia as actin–cortactin rich aggregates. The yellow arrowhead points to high numbers of invadopodia associated with the cell body. (E) Effect of kindlin2-GFP phosphomimetic mutants on invadopodia formation in MDA-MB-231 cells invading globular collagen matrix at the absence of serum. Cells were cultured on collagen for 3 h, fixed, and immunolabeled for actin and cortactin to identify invadopodia as actin–cortactin-rich aggregates. Yellow arrowheads indicate invadopodia on the cell body. N/T, not transfected; N/S, nonspecific; wt, wild type. *, P < 0.05; **, P < 0.001. Bars, 10 µm.