Paxillin- and Hic-5–specific functions in invadosome formation and activity. (A) Representative Western blot of cell lysates from MEF-SrcY527F cells treated with control, paxillin, Hic-5, or both paxillin and Hic-5 siRNA (left). Quantification of paxillin and Hic-5 revealed that the acute depletion of each protein led to an adaptation response by increasing the protein levels of its related protein (n = 5; middle). Quantification by quantitative real-time PCR showed that the siRNA treatment affected only the targeted mRNA without affecting the counteract protein mRNA (n = 3; right). (B) MEF-SrcY527F cells treated with control, paxillin, Hic-5, or paxillin and Hic-5 siRNA were spread on fibronectin-coated glass coverslips and stained with anti-paxillin antibody (green) and F-actin (red). (C) Quantification of the normalized number of invadosome rings per cell revealed that paxillin silencing decreased the number of invadosome rings per cell, whereas silencing of Hic-5 significantly increased it (n = 3, 300 cells counted per condition). (D) Quantification of the mean degradation area per cell revealed that all siRNA treatments decreased the degradation activity compared with the control (n = 3; 200 cells counted per condition). **, P < 0.01. (E) Quantification of the GFP-actin characteristic time of recovery showed that the depletion of both paxillin and Hic-5 did not affect actin mobility in invadosomes (n = 3). (F) Representative images extracted from a time series of MEF-SrcY527F cells stably expressing LifeAct-GFP and transfected with specific siRNAs (control, paxillin, or Hic-5). Paxillin depletion resulted in very thick and hyperstable invadosome rings, whereas Hic-5-knockdown cells exhibited normal dynamics, as in the control siRNA-treated cells. All graphs presented as means ± SD. Differences with a probability level P < 0.05 were considered significant in one-way ANOVA. Bars: (A) 6 µm; (F) 15 µm.