CXCR4 overexpressed on cAMP-stimulated CD34⁺ progenitors is functional. (A) Transendothelial migration of dbcAMP (cAMP)-treated cells. CD34⁺ progenitors were incubated for 24 hours with 500 μM cAMP or left untreated (-), labeled with CFSE, and allowed to migrate either spontaneously (spont) or toward SDF-1 (directional) through BMEC-coated transwells. Data represent the percentages of migrated cells (mean ± SD of 2 independent experiments). (B) Adhesion to BM cells after cAMP treatment. Cells treated for 24 hours with 500 μM cAMP or left untreated (CTRL) were labeled with CFSE and allowed to adhere for 40 minutes to MS-5 cell layer (mean ± SD of at least 2 independent experiments, each in triplicate samples). Where indicated, cAMP-treated cells were preincubated with 10 μM PKCζ PS (PSζ), 10 μg/mL VLA-4 blocking mAb (αVLA-4), or IgG1 control. (C) Homing efficiency of cAMP-treated cells. CB or MPBL CD34⁺ cells were treated for 24 hours with 500 μM cAMP or left untreated (CTRL) and injected intravenously into the NOD/SCIDB2m^null mice. Where indicated, cAMP-treated cells were incubated for 30 minutes before injection with 10 μM PSζ or cultured in the presence of 25 μM NSC23766 (Rac1 inh). BM and spleen of recipient mice were analyzed for the presence of human cells (mean ± SD of at least 3 independent experiments, 2 to 3 mice per treatment in each experiment; ^*P < .05 relative to other treatments). Representative flow cytometry analyses are shown on the right. IgG indicates samples labeled with the isotype control Ab. Number of CD45⁺ human cells per 10⁶ acquired is indicated in each case.