cAMP-stimulated CXCR4 expression in CD34⁺ hematopoietic progenitors is Rap1 dependent. (A) Membranal CXCR4 expression on human MPBL or CB CD34⁺ cells treated for 24 hours with 500 μM dbcAMP (cAMP) and 10 nM PGE2, untreated, or vehicle EtOH-treated as control (CTRL), indirectly immunolabeled with anti-human CXCR4 mAb. Results are shown as mean ± SD. For each cell source, 20 cAMP and 5 PGE2 independent experiments were conducted. P values indicate statistically significant differences compared with CTRL. Representative flow cytometry analysis is shown at the bottom. IgG indicates secondary Ab-only labeled samples. (B) Representative analysis of activated Rap1 (Rap1-GTP) status in G2 cells stimulated for 24 hours with 500 μM cAMP or left untreated (CTRL). (C) Effect of Spa1GFP overexpression on membranal CXCR4 level in MPBL CD34⁺ cells incubated 18 hours after transfection and left untreated (CTRL) or treated with 500 μM cAMP. Results are shown as fold change compared with the CXCR4 intensity in GFP-only transfected samples taken as 1 (mean ± SD of 4 independent experiments). (D) Time-lapse analysis of membranal CXCR4 expression in G2 cells treated with 500 μM cAMP for the indicated time periods. Results (mean ± SD of 3 independent experiments) are expressed as fold change compared with CXCR4 intensity of untreated counterparts.