CXCR7 and CXCR4 mediate the CXCL12-induced Akt phosphorylation in PB CD34⁺cells through β-arrestins. (A) PB CD34⁺ cells were incubated or not in serum- and cytokine-free medium supplemented with CXCL12 (0.5 ng/mL) for 45 minutes. β-arrestin2 and CXCR7 localization were analyzed using a TC5 SP5 confocal microscope (Leica, Wetzlar, Germany) after labeling with specific antibodies. The scale bar represents 10 µm.. (B) PB CD34⁺ cells were incubated in serum- and cytokine-free medium supplemented with CXCL12 (0.5 ng/mL) and blocking anti-CXCR4 antibody or CXCR7 inhibitor (CCX733) for 45 minutes. The translocation of β-arrestin2 to the nucleus was analyzed with a TC5 SP5 confocal microscope (Leica) after labeling with specific antibody. Histograms show the ratio of β-arrestin2 nuclear translocated cells. Asterisks indicate significance between conditions (*P ≤ .05, **P ≤ .01). The scale bar represents 10 µm. (C) PB CD34⁺ cells were stimulated 15 minutes in a serum- and cytokine-free StemαA medium in the presence or absence of CXCL12 (0.5 ng/mL), CCX704, or CCX771 compounds, blocking anti-CXCR4 antibody, or isotype control IgG. After phospho-Akt-Ser473 labeling, the percentages of expressing cells were determined by flow cytometry by comparison with the isotype control profile. The histogram shows the percentage of phospho-Akt–expressing cells as mean ± SD (n = 3 to 4). Deltas indicate significance between conditions (ΔΔP ≤ .01, ΔΔΔP ≤ .001). (D) Scramble or β-arrestin2 siRNA–transfected CD34⁺ cells were incubated with CXCL12 (0.5 ng/mL) for 5, 10, 15, and 30 minutes. Western blot analysis was then done and the membrane was immunoblotted with specific antibodies against pAkt, total Akt, actin, and β-arrestin2. The histogram shows the percentage of β-arrestin2–expressing CD34⁺ cells after scramble or β-arrestin2 siRNA transfection. The asterisk indicates significance between conditions (*P ≤ .05).