Fig. 7. Podosome loss induced by PGE₂, EP agonists and elevated cAMP is mediated by Rho kinase. (A) Rho-kinase inhibition blocks PGE₂- and EP-agonist-induced podosome dissolution. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE₂ (for 5 minutes), butaprost (buta, 2 µM for 15 minutes) or PGE₁-OH (5 µg/ml for 30 minutes), in the presence or absence of Y27632 (20 µM for 1 hour), or were treated with Y27632 alone, and stained with anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was determined in seven images per condition per experiment and an average (with s.e.m.) of three experiments is shown. (B) IBMX and IBMX/forskolin-induced podosome dissolution is efficiently blocked by Y27632. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with IBMX (10 µM for 5 minutes) or IBMX with forskolin (IBMX/fors, both 10 µM for 5 minutes) in the presence or absence of Y27632 (20 µM for 1 hour), or with Y27632 alone, and stained with anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was counted in seven images per condition per experiment and an average (with s.e.m.) of three experiments is shown. Asterisks indicate significant differences (P<0.05). (C) Rho-kinase inhibition blocks podosome loss in response to PGE₂, EP2/EP4 agonists or cAMP elevation. Representative images are depicted.