Figure 7
α1-AR regulate internalization of CXCR4 in human vascular smooth muscle cells and CXCR4-mediated chemotaxis. (a) hVSMC were labeled for 1 hour at 4 °C with rabbit anti-CXCR4 (ACR-014, left) or FITC-conjugated-anti-CXCR4 (12G5, right), and then treated with vehicle, 100 nM CXCL12 or 10 μM PE for 20 min at 37 °C. Cells stained with anti-CXCR4 (ACR-014) were permeabilized with TX-100 and stained with Alexa 488-conjugated anti-rabbit. Fluorescence microscopy images are representative of n = 3 independent experiments. Scale bar: 20 μm. (b) Representative PLA images for the detection of individual receptors and receptor-receptor interactions in hVSMC. Cells were treated with vehicle or PE (1μM) for 15 min at 37 °C. Ctrl.: Omission of one primary antibody. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride signals. Scale bars = 10 μm. (c) Quantification of PLA signals, as in b. N = 3 with n = 10 images per condition and experiment. *p < 0.05 vs. ctrl. (d) Migration of hVSMC towards PE (hatched bars), CXCL12 (10 nM, open bar) and combinations of both (grey bars). Dark grey bars: migration of hVSMC in the presence of various concentrations of PE towards 10 nM CXCL12. Light grey bars: migration of hVSMC towards 10 nM CXCL12 plus various concentrations of PE (insert shows the dose-response curve). N = 4 independent experiments. (e) hVSMC were pre-treated with 10 μM phentolamine, 10 μM AMD3100, or vehicle for 15 min at room temperature, followed by migration towards 1 μM PE, 10 nM CXCL12, or both. N = 4 independent experiments. *p < 0.05 vs. vehicle.
