Molecular mechanisms for AM1241/CB2-induced HSPC migration and mobilization. (A) Effect of distinct inhibitors in AM1241-induced migration of human CD34⁺ cells. A total of 1 × 10⁴ cells were placed in the upper well of transwell inserts, and AM1241 (1μM) or vehicle control was added to the bottom compartments. The system was then incubated for 4 hours at 37°C, and the cells that migrated to the lower compartment were counted. As indicated, the following inhibitors were added to CD34⁺ cells 45 minutes at 37°C in a CO₂ incubator before AM1241 treatment: 10μM LY294002 (LY), 25μM U0126 (U), 100 ng/mL of pertussis toxin (PTX), or 1μM AM630. Data are mean ± SD (n = 4). *P < .01 is statistically significant compared with control cells. (B) Effects of AM1241 on Rac1-GTPase activity. Human MNCs were either untreated or pretreated with 100 ng/mL of PTX for 1 hour followed by stimulation with AM1241 (1μM) for 4 minutes. Rac1-GTP levels were then measured using Rac1-GTPase pull-down assay. The results are normalized to the density of total Rac1 band in the corresponding samples as analyzed by GST pull-down assay, followed by Western blot analysis (n = 3). *P < .01, compared with control untreated cells. (C-D) Effect of CP55940 on the CXCL12 (SDF-1)–induced migration of CD34⁺ cells (C) and murine LSK cells (D). Cells were placed in the upper chamber with or without 1μM AMD3100 (CXCR4 antagonist) or 10nM CP55940. A total of 100 ng/mL CXCL12 was placed the lower chamber as indicated. Cells were allowed to migrate for 4 hours, and then cells in the lower chamber were counted. Data are mean ± SD (n = 12). P < .01 compared with control. (E) Synergistic effects of AM1241 and AMD3100 treatment. Mice were injected intraperitoneally with AM1241 at a dose of 10 mg/kg once daily for 4 consecutive days. Twenty-four hours after the last injection of AM1241, mice were injected intraperitoneally with AMD3100 at a dose of 5 mg/kg. One hour after injection of AMD3100, mice were killed, their peripheral blood was collected, and MNCs were isolated and seeded (1 × 10⁵ cells per dish). Ten days after seeding, colonies were counted under a light microscope. Data are mean ± SD (n = 6). *P < .05 vs vehicle control. **P < .05 vs each drug alone. (F-G) The affinity binding of AM1241 and SDF-1α to CXCR4. The 293T cells and MDA-MB-231 cells were grown to a density of 75% in 96-well puncher plates. To assign absolute affinity of each ligand for CXCR4, a competitive displacement assay was used. To avoid internalization of the radioligand resulting from constitutive endocytosis, live cell binding was performed at 4°C. Well contents were counted on a model 1470 Wallac Wizard (PerkinElmer Life and Analytical Sciences) detector γ-counter.