Hic-5 interacts specifically with IQGAP1 and controls its recruitments to invadosomes. (A) The specific coimmunoprecipitation of Hic-5-WT-Flag and IQGAP1-GFP, transfected in HEK 293 cells, showed the specific interaction of these two proteins. (B) Immunoprecipitation of endogenous IQGAP1 or Hic-5 in MEF-SrcY527F cells is associated with the respective specific coimmunoprecipitation of Hic-5 and IQGAP1, in comparison to IgG control. (C) Confocal imaging of MEF-SrcY527F cells expressing Hic-5-Flag and IQGAP1-GFP showed their colocalization. (D) TIRF imaging of MEF-SrcY527F cells shows IQGAP1-GFP localization in invadosome revealed by RFP-cortactin (left). Confocal imaging of endogenous IQGAP1 also revealed its accumulation in invadosomes (right). (E) Quantitative confocal imaging showed that Hic-5 silencing is associated with a decrease in endogenous IQGAP1 recruitment in invadosomes (left; n = 3; 60 invadosomes per condition). Moreover, MEF-SrcY527F Hic-5^−/− cells presented a representative decrease in IQGAP1 recruitment in invadosomes, in comparison with MEF-SrcY527F Hic-5^−/− cells reexpressing Hic-5-WT-Flag (n = 3; 55 invadosomes per condition). *, P < 0.05; **, P < 0.01. (F) Western blot of cell lysates from MEF-SrcY527F cells treated with control or IQGAP1 siRNA (top). Quantitative confocal imaging shows that IQGAP1 silencing decreases plasma membrane accumulation of MT1-MMP in invadosomes (n = 3; 60 to 70 invadosomes per condition; bottom). ***, P < 0.001; **, P < 0.01. (G) Quantitative confocal imaging shows that Hic-5 silencing mimics IQGAP1 silencing in MT1-MMP accumulation at the surface of invadosomes (n = 3; 50 invadosomes per condition). All graphs presented as means ± SD. Differences with a probability level P < 0.05 were considered significant in one-way ANOVA. Bars: (C–E) 8 µm; (F and G) 5 µm.