FIGURE 5.

Src kinase activity is required for podosome formation and for filamin A localization to podosomes. MDMs plated on fibrinogen (A) and treated with SU6656 (B) were stained for hFLNa and F-actin, before acquisition of confocal micrograph series (a and b, respectively) (z-step = 0.1 μm) (scale bar, 10 μm). a′ and b′ show the average of the F-actin and FLNa fluorescence staining of at least 100 podosomes from control and SU6656-treated cells (scale bar = 1 μm). a″ and b″ show fluorescence intensity profiles of the averaged podosomes along the white dashed line in (a′) and (b′), respectively. C, quantification of human MDMs with podosomes or podosome rosettes when seeded on coverslips that were either uncoated or coated with fibrinogen (Fg) and treated with the Src inhibitor SU6656 (mean ± S.D. of three independent experiments). D, RAW264.7 macrophages or stably expressing a shRNA against mouse Hck were (or not) transiently transfected with a human Hck-GFP coding vector (to rescue Hck) and were transfected transiently with LifeAct-mCherry coding vector to stain F-actin. Podosome lifespans, measured by time-lapse microscopy, are plotted for each cells type (mean ± S.D. of three independent experiments, five to 10 podosomes analyzed per cell in at least three cells per experiment).