cAMP-induced increase in CXCR4 expression depends on PKCζ activity. Membranal CXCR4 expression in CB and MPBL CD34⁺ (A) or B1 (B) cells, incubated for 24 hours with 500 μM dbcAMP (cAMP), 2 μM cholesterol sulfate (Choles Sulfate), or left untreated (-). Where indicated, 10 μM PS of PKCζ (PSζ), PKCϵ (PSϵ), PSα/β (PSα/β), or 25 μM NSC23766 (Rac1 inh) were applied. Flow cytometry analysis data are shown in arbitrary units (AU) as mean ± SD; 10 independent experiments were conducted for PSζ, and 6 were conducted for Rac1 inh. (C) Increase in membranal and intracellular CXCR4 labeling in G2 cells incubated for 24 hours in the presence of 500 μM cAMP compared with untreated cells (CTRL) taken as 1. Flow cytometry analysis data (mean ± SD of 3 independent experiments) are shown. (D) Decreased internalization rate of anti-CXCR4-PE mAb in G2 cells treated for 24 hours with cAMP compared with CTRL. Results are mean ± SD of 3 independent experiments. P < .02; statistically significant differences compared with CTRL at each time point. Increased recovery after SDF-1-induced receptor internalization is shown in the inset. Mean fluorescence intensity (MFI) is calculated as percentage of CXCR4 expression in CTRL or cAMP-treated cells before the application of SDF-1 (original MFI). (E) Relative CXCR4 expression in G2 cells either untreated (-) or stimulated for 6 hours with 500 μM cAMP in the absence or presence of 10 μM PSζ, as analyzed by real-time RT-PCR. Data are expressed in AU as a ratio of CXCR4 and β-actin mRNA level (mean ± SD of 3 independent experiments). (F) Changes in CREB phosphorylation (P-CREB) in MPBL CD34⁺ progenitors stimulated for 1 hour with 500 μM cAMP (mean ± SD of 3 independent experiments). Where specified, cells were pretreated with either 10 μM PSζ or 18 μM NF-κBSN50 (NF-κB inh). Representative flow cytometry analysis is shown. IgG indicates secondary Ab-labeled cells; CTRL, untreated samples.