Fig.4. P2Y₂R expression was increased in THP-1 human monocytes in hypoxia, and P2Y₂R activation by ATP or UTP induced THP-1 migration and the release of MMPs.

A) THP-1 human monocytes were incubated in hypoxia for 24 h, and then P2Y₂R mRNA was analyzed by RT-PCR. Results were confirmed by repeated experiments. B) THP-1 cells were transfected with control siRNA or P2Y₂R siRNA (100 nM), and the efficiency of siRNA transfection was confirmed by RT-PCR. C-E) Control siRNA- or P2Y₂R siRNA-transfected THP-1 cells (2 × 10⁵ cells) were added to 24-well cell culture inserts, and supernatants from MDA-MB-231 cells exposed to hypoxia in the presence or absence of apyrase (5 U/ml), an enzyme that rapidly hydrolyzes extracellular nucleotide 5′-diphosphates and triphosphates, for 8 h (D) or media containing ATP (10 μM) or UTP (10 μM) (E) were added to the lower chamber of the Transwell. After incubation for 6 h in a 37°C cell culture incubator, the cells that had migrated through the insert membranes were stained with DAPI and counted under a fluorescence microscope. Values represent the means ± SEM of 3 independent experiments (compared with the control, **P < 0.01, *P < 0.05) (scale bar: 50μM). F) Control siRNA- or P2Y₂R siRNA-transfected THP-1 cells were treated with ATP (10 μM) or UTP (10 μM) for 24 h, and the activity of MMP-2 and MMP-9 in the CM was evaluated by gelatin zymography as described in the Methods.