Membrane and intracytoplasmic CXCR4 expression in migrating and nonmigrating HSCs in response to CXCL12 and UTP. (A) Percentages of CXCR4⁺ cells were evaluated in migrating and nonmigrating CD34⁺ HSCs, collected from the lower and upper chamber of transwell plates, respectively. Among CD34⁺ cells migrating toward a CXCL12 gradient, we found the down-regulation of membrane CXCR4 compared with nonmigrating cells. However, when UTP was added to the upper chamber, no difference in the percentage of CXCR4⁺ cells was detected between migrating and nonmigrating HSCs. Intracytoplasmic staining of CXCR4 (B) demonstrated that the down-regulation of cell membrane CXCR4 is due to its internalization, which is inhibited by addition of UTP. Results were from 3 independent experiments and are shown as mean ± SEM. (C) In inhibition assays, human CD34⁺ HSCs were preincubated with PTX (1 μg/mL) as described in “Materials and methods.” PTX treatment almost completely abrogated spontaneous migration of UTP-treated cells and CXCL12-dependent chemotaxis. Furthermore, the CXCL12-induced/UTP-supported migration was completely blocked by treatment with PTX (93% inhibition; P < .05). Cells treated with anti-CXCR4 mAb (50 μM/mL) showed a decreased response to CXCL12 gradients, even when simultaneously cultured with UTP. Results were from 4 independent experiments and are shown as mean ± SEM. (D) Cytosolic Ca²⁺ concentration changes induced by UTP and CXCL12. Cells were loaded with fura-2/AM as reported in “Materials and methods.” UTP concentration was 10 μM, while CXCL12 concentration was 150 ng/mL. The arrow indicates addition of the stimuli. UTP (dashed line), CXCL12 (dotted line), and UTP + CXCL12 (continuous line). Results were from 3 independent experiments and are shown as mean ± SEM. *P values less than .05.