Expression of CXCR7 increases the in vitro chemotactic response of MTLn3 CXCR4 cells to CXCL12. (a) RT-PCR of RNA isolated from the indicated transductants using primers specific for either rat GAPDH, rat CXCR4, human CXCR4, or both rat and human CXCR7. (b) FACS analysis of the transductants. Representative FACS plots show receptor expression at the cell membrane: isotype control mouse IgG (grey shaded peaks), anti-CXCR4 antibody (solid lines, MAB172) and anti-CXCR7 antibody (dashed lines, 11G8). (c) Chemotaxis of transduced cell lines to CXCL12. Cells were allowed to chemotax for four hours at 37°C in a microchemotaxis chamber. Total number of cells per well are reported (11 to 33 wells were counted per condition). Comparison of MTLn3 CXCR4 chemotaxis to CXCL12 with that of MTLn3 JP cells shows a statistically significant increase at 0.25 nM, 1 nM, 10 nM, and 100 nM with a P value less than 0.005 as determined by t-test, and P = 0.079 at 0.05 nM CXCL12. Comparison of the chemotaxis of the double expressors, CXCR4-CXCR7, with that of MTLn3 JP cells show similarly statistically significant differences as determined by t-test, with P < 0.05 at 0.05 nM CXCL12 and P < 0.005 at the other concentrations. Statistically significant differences between MTLn3-CXCR4 and MTLn3 CXCR4-CXCR7 are indicated in the figure with P < 0.05 indicated by * and P < 0.005 indicated by **. (d) Chemotaxis of MTLn3 CXCR4 and MTLn3 CXCR4-CXCR7 cells to 1 nM CXCL12 with or without 100 nM AMD3100 (6 to 22 wells were counted per condition), P < 0.005 is represented by **. (e) Chemotaxis of MTLn3 CXCR4 and MTLn3 CXCR4-CXCR7 cells to 1 nM CXCL12 with or without 10 nM I-TAC (five to seven wells were counted per condition). (f) MTLn3 CXCR4-CXCR7 chemotaxis to 1 nM CXCL12 in the presence of vehicle DMSO, CCX771, or CCX733 (11 to 15 wells were counted per condition). Means and SEMs are shown.