The involvement of PI2K, ERK1/2, and Rac in SDF-1-induced migration. (A) CD34⁺ cells from healthy donors were pretreated with 10 μM U0126, 10 μM LY294002, or 40 μM NSC23766 for 30 minutes, and applied to the upper compartment of a microchamber transwell system with 5-μm pores. Migration was induced by 100 ng/mL SDF-1 for 4 hours at 37°C after which the transmigrated CD34⁺ cells were harvested from the lower chamber and counted by FACS analysis. The assay was done in duplicate, and the ratio between transmigrated CD34⁺ cells and the total amount of CD34⁺ cells added to the upper well was calculated. The number of cells migrated in the presence of inhibitors is shown as a percentage of the number of cells migrated in the control samples. The means of 6 individual experiments are shown. (B,C) Normal CD34⁺ cells were pretreated with 10 μM U0126 or 10 μM LY294002 for 30 minutes where indicated and subsequently stimulated with 100 ng/mL SDF-1. PKB and ERK1/2 activation were detected by Western blotting, using antibodies against phosphorylated PKB or ERK1/2 (top). The blots were reprobed with total ERK1/2 antibody, to control for equal loading. Three independent experiments were performed, and one representative experiment is shown. (D) Normal CD34⁺ cells were stimulated with 100 ng/mL SDF-1 for the indicated time, with or without pretreatment of cells with 40 μM NCS23766 for 30 minutes. Activated Rac was precipitated using GST-PAK-CRIB and visualized by Western blotting with Rac antibodies. One representative example of 3 independent experiments is shown. (E) HL60 cells that were transduced with mock empty vector, Rac1V12 or Rac1N17 were stimulated with 100 ng/mL SDF-1. Activated Rac was precipitated using GST-PAK-CRIB and visualized by Western blotting with Rac antibodies. One representative example of 3 independent experiments is shown. (F) HL60 cells that were transduced with mock empty vector, Rac1V12, or Rac1N17 were applied to the upper compartment of a microchamber transwell system with 8 μm pores. Migration was induced by 100 ng/mL SDF-1 for 4 hours at 37°C after which the transmigrated cells were harvested from the lower chamber and counted by FACS analysis. The assay was done in duplicate, and the ratio between transmigrated HL60 cells and the total amount of cells added to the upper well was calculated. The means of 5 experiments are shown.