Fig. 3

CCR2 and GPCR βγ activity is required for MCP-1-mediated migration. a Surface CCR2 re-localises after MCP-1 treatment. Immunofluorescent staining of non-permeabilised serum-starved BMSCs, following the indicated treatments at the indicated times. CCR2 (red). α-tubulin (green). DNA stained with DAPI (blue). Scale bar = 10 μm. b Immunoblotting of BMSC whole cell lysates for CCR2 and β-actin following MCP-1 treatment; 100 μg of total cell extract/lane. Representative blot of three separate independent experiments is shown. c Kinetics of Pericentrin-1 re-localisation after MCP-1 treatment. Immunofluorescent staining of permeabilised serum-starved BMSCs. CCR2 (red) and Pericentrin-1 (green). DNA stained with DAPI (blue). Scale bar = 10 μm. d Serum-starved BMSCs pre-treated with 10 μM RS102895 (βγ GPCR subunit inhibitor) and stained after exposure to MCP-1. CCR2 (red). α-tubulin (green). DNA stained with DAPI (blue). Scale bar = 10 μm. e 24-h treatment with 10 μM RS102895 combined with serum starvation inhibits MCP-1-mediated BMSC migration in 3D. Graph represents three separate independent experiments. Data are presented as mean ± SD. f MCP-1-mediated Pericentrin-1 re-localisation is inhibited by 24-h pre-treatment with 10 μM MPS-Phos (GPCR βγ inhibitor) and SFM. Permeabilised BMSCs stained with CCR2 (red) and α-tubulin (green). DNA stained with DAPI (blue). Scale bar = 10 μm. g Combination of serum-starved BMSCs with a GPCR βγ inhibitor (10 μM MPS-Phos) for 24 h significantly reduced MCP-1-mediated migration in 3D. Graph represents three separate independent experiments. Data are presented as mean ± SD. A P value of less than 0.05 (*) was deemed significant. 3D three dimensions, BMSC bone marrow-derived stromal cell, CCR2 chemokine (C motif) receptor 2, DAPI 4′,6-diamidino-2-phenylindole, GPCR G protein-coupled receptor, MCP-1 monocyte chemoattractant protein 1, MPS-Phos MPS-phosducin-like protein C terminus, SD standard deviation, SFM serum-free medium