Fig. 5. HL-60 cells form podosomes upon PMA stimulation and respond to PGE₂. (A,B) HL-60 cells form podosomes upon stimulation with PMA, which dissolve after stimulation with PGE₂ or an EP4 agonist. HL-60 cells seeded on FN-coated coverslips were left untreated or were stimulated with PMA for 24 hours and then left untreated or were further stimulated with PGE₂ for 5 minutes. Cells were then stained with an anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). (A) Representative images are depicted. (B) 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. (C) HL-60 cells show expression of EP2 and EP4, whereas EP1 and EP3 receptors are nearly undetectable, as determined by RT-PCR. Asterisks indicate significant differences (P<0.05).