Fig. 3. PGE₂-induced podosome loss is mediated by the EP2 and EP4 receptors. (A) iDCs and mDCs express the EP2 and EP4 receptors. On iDCs and mDCs, an RT-PCR for the PGE₂ receptors EP1-EP4 was performed. Controls include β-actin (actin) and minus RT (–). A specific product is detected for EP2 and EP4 but not for EP1 and EP3 receptors. (B) EP2 and EP4 antagonists block PGE₂-induced podosome dissolution. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE₂ in the presence or absence of AH6809 (5 µM for 30 minutes) or AH23848 (50 µM for 30 minutes), or with AH6809 or AH23848 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 four experiments is shown. (C) EP2 and EP4 agonists and cAMP elevation mimic PGE₂ in inducing podosome dissolution. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE₂, butaprost (buta, 2 µM for 15 minutes), PGE₁-OH (5 µg/ml for 30 minutes), sulprostone (sulp, 2 µM for 15 minutes), IBMX (10 µM for 15 minutes) or IBMX with forskolin (IBMX/fors, 10 µM for 15 minutes), and stained for vinculin and 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 for the EP agonists (buta, PGE₁-OH and sulp) and seven experiments for cAMP elevation (IBMX and fors) is shown. Asterisks indicate significant differences (P<0.05).