Functional redundancy of paxillin and Hic-5 in invadosome formation. (A) Paxillin depletion in MEF-SrcY527 cells does not affect invadosome formation. The reexpression of either pax-WT-Flag or Hic-5-WT-Flag reinduces the formation of invadosomes easily recognized by their ring shape (arrowheads). (B) Western blot analysis of pax^−/− cells expressing or not expressing Hic-5 or pax-WT-Flag. shRNA treatment efficiently decreased the endogenous protein levels of Hic-5, whereas expression of pax-WT-Flag reinforced the effects of Hic-5 silencing. (C) Quantification of the percentage of cells forming invadosomes in MEF-SrcY527F pax^−/− cells rescued or not rescued with Hic-5 or pax-WT-Flag shows the redundancy of both proteins (n = 4; 500 cells per condition). ***, P < 0.001. (D) Representative analysis of the fluorescence intensity profile of paxillin-GFP or Hic-5-GFP and LifeAct-RFP over the yellow lines going through the invadosome revealed the same distribution of both proteins as a crescent-shaped gradient from the inner to the outer rims (n = 4). au, arbitrary unit. (E) FRAP experiments (n = 3) performed on MEF-SrcY527F cells expressing either paxillin-GFP or Hic-5-GFP revealed that both proteins presented the same mobility in invadosomes. Red rectangles show where the photobleaching is induced. All graphs presented as means ± SD. Differences with a probability level P < 0.05 were considered significant in one-way ANOVA. Bars: (A) 10 µm; (D) 2 µm; (E) 4 µm.