FIGURE 5.

Podosome formation as physiological readout for ADAM17 function. (A) Podosomes were visualized by immunofluorescence stainings in LPS-stimulated M-MØ with ADAM17-deficient background (ADAM17^ex/ex) either expressing empty vector (mock), ADAM17 wt, or cytoplasmic deletion mutant ADAM17 Δ700. Podosomes were visualized by staining F-actin with Alexa Fluor 488–labeled phalloidin (green), and cell membranes were highlighted by using a CellMask staining (red) to clearly distinguish individual cells for quantification. Scale bar, 10 μm. (B) As a second marker for podosomes, vinculin, a component of the characteristic podosome ring structure, was stained using a specific primary Ab (green). F-actin staining is shown in red. Scale bar, 10 μm; insets: 1 μm. Quantification of podosome staining in mock, wt, and Δ700-reconstituted cells expressed as podosomes per cell (C) and podosome density (podosomes/100 μm²) (D). (E) For analysis of podosome number and density, the cell area of M-MØ was determined by a cell-surrounding white line [as shown in (A) and (B)]. No statistical difference was found between the cell area of mock, ADAM17 wt, and ADAM17 Δ700–reconstituted M-MØ (n = 3 × 30; each time, 30 cells were analyzed in three independent cell differentiations). A one-way ANOVA followed by a Tukey multiple comparison test was applied. *p < 0.05, ***p < 0.005.