Endothelial P2Y6 receptor up-regulation after TNF-α stimulation. HCAECs (A-B) or HMEC-1 cells (C-D) were exposed to TNF-α for the indicated time periods or with indicated doses. P2Y6 receptor transcript was determined by real-time RT-PCR. Data were calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean ± SD fold change compared with controls (without TNF-α) (n = 3-4). (E) HMEC-1 cells were exposed to LPS, IL-1α, and TNF-α for 24 hours. (F) HCAECs were grown to confluence on cover glasses and exposed to 10 ng/mL TNF-α for 24 hours. Cell layers were stained with antibodies specific for human P2Y6 receptor and Alexa Fluor 488–coupled secondary antibody (green) or isotype controls and Alexa Fluor 488–coupled secondary antibody or Alexa Fluor 488–coupled secondary antibody only. DAPI (4′,6-diamidino-2-phenylindole) was used as nuclear counterstain (blue). Slides were kept on ice before image acquisition. Probes were analyzed by confocal microscopy with the use of Zeiss Laser Scanning Microscope LSM 710 and the Plan-Apochromat 63×/1.40 Oil Dic M27 objective lens with oil immersion. Zen software was used for acquisition and image processing (γ adjustment) applied equally to all images. One representative image of 3 is displayed. (G) HMEC-1 cells were exposed to TNF-α for the indicated time periods, and P2Y6 receptor protein was determined by Western blotting. The same blot was stripped and reprobed for human β-actin as a control for protein loading. One of 3 representative Western blots is displayed.
