ADAM17 is required for TLR-signaling induced podosome disassembly. BMDC were cultured from ADAM17^+/+, ADAM17^+/fl, or ADAM17^fl/fl mice. Lysates from these were immunoblotted with antibodies against ADAM17 or actin (loading control). (B) TNF-α, released by BMDC during treatment with 50 ng/ml LPS or 100 ng/ml Pam3CSK for 2 h, was measured by ELISA. (C) ADAM17^+/+, ADAM17^+/fl, or ADAM17^fl/fl BMDC were stimulated with 50 ng/ml LPS or 100 ng/ml Pam3CSK for 30 min, then assessed, after staining with TRITC-phalloidin, for the presence of podosomes. (D) Confocal images of the cells quantitated in C confirm that the LPS- or Pam3CSK-stimulated podosome loss observed in ADAM17^+/+ cells is inhibited in ADAM17^fl/fl cells. (E) The percent inhibition of podosome loss versus the percent inhibition of TNF-α shedding (relative to +/+ controls) for experiments performed on BMDC (squares) or SDC (circles), which had been treated with LPS (closed symbols) or Pam3CSK (open symbols), as before, from five ADAM17^fl/fl mice. The Pearson coefficient for the data is 0.76. (F) Immunoblots of lysates from uninfected BMDC or BMDC infected with retrovirus expressing either GFP marker alone or GFP and ADAM17 show that expression of ADAM17 can be restored in ADAM17^fl/fl cells. ADAM17^+/+ or ADAM17^fl/fl BMDC infected with GFP or ADAM17 retrovirus were left untreated or stimulated with 50 ng/ml LPS for 30 min, then stained with TRITC-phalloidin. GFP⁺ (infected) cells from each condition were scored for the presence of podosomes (G), and representative confocal images of LPS-treated cells are shown (H). Bars, 20 μm.