Figure 2

A. Concentration-dependent inhibition of LD78β- and SDF-1-induced intracellular calcium mobilization by SCH-C and AMD3100 in CCR5- and CXCR4-transfected U87.CD4 cells and in the double-transfected U87.CD4.CCR5.CXCR4 cells. The Fluo-3 loaded cells were preincubated for 10 minutes with SCH-C at 200, 40, 8 and 1.6 ng/ml or AMD3100 at 1000, 200, 40 and 8 ng/ml. Then U87.CD4.CCR5 cells were stimulated with LD78β at 100 ng/ml (upper left graph) and the U87.CD4.CXCR4 cells were stimulated with SDF-1 at 20 ng/ml (upper right graph). U87.CD4.CCR5.CXCR4 cells were stimulated with either LD78β at 100 ng/ml (lower left graph) or SDF-1 at 20 ng/ml (lower right graph). The transient increase in intracellular calcium concentration was recorded by monitoring the change in green fluorescence intensity of the cells (y-axis) as function of time (x-axis) using the Fluorometric Imaging Plate Reader (FLIPR). Each data point represents the average value of the fluorescence measured in quadruplicate. The data of one representative experiment of four are shown. B. LD78β- and SDF-1-induced intracellular calcium mobilization and blocking by SCH-C and AMD3100. Fluo-3 loaded cells were preincubated with a 1:1 combination of SCH-C and AMD3100 at 1000 ng/ml for 10 minutes, after which LD78β (100 ng/ml) and SDF-1 (20 ng/ml) were added sequentially at timepoints 20 seconds and 222 seconds respectively (arrows). The transient increase in intracellular calcium concentration was recorded by monitoring the change in green fluorescence intensity of the cells (y-axis) as function of time (x-axis) using the FLIPR. Each data point represents the average value of the fluorescence measured in quadruplicate. The data of one representative experiment out of four are shown.